Methods and compositions for preventing and treating male erectile dysfunction and female sexual arousal disorder

ABSTRACT

The invention provides a method for preventing or treating male erectile dysfunction or female sexual arousal disorder by administering an effective amount of one or more factors from a group of factors including vascular endothelial growth factor, brain-derived neurotrophic factor, basic fibroblast growth factor, neurotrophin-3, neurotrophin-4, or angiopoietin-1, wherein the factor is a full length protein or a nucleic acid encoding the factor, or a functional derivative or fragment thereof, or an agent that enhances production and/or male erection or female sexual arousal stimulating function of the factor(s). Combinations, kits, and combinatorial methods are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation-in-part of U.S. patentapplication Ser. No. 09/909,544, filed Jul. 19, 2001, now pending, whichclaims the benefit of the priority date of U.S. provisional patentapplication Serial No. 60/220,031, filed Jul. 21, 2000, under 35 U.S.C.§119(e). The disclosure of the above-described applications areincorporated herein by reference in their entirety

STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH

[0002] This invention is supported in part by Grant No. DK45370 andDK51374 of the National Institutes of Health. The United Statesgovernment may have certain rights in this invention.

FIELD OF THE INVENTION

[0003] This invention relates generally to the field of urology. Inparticular, the invention provides a method for preventing or treatingmale erectile dysfunction or female sexual arousal disorder in a mammalin need of such treatment, comprising administering an effective amountof a factor from a group of factors including vascular endothelialgrowth factor (VEGF), brain-derived neurotrophic factor (BDNF), basicfibroblast growth factor (bFGF), platelet-derived growth factor (PDGF),neurotrophin-3 (NT-3), neurotrophin-4 (NT-4), or angiopoietin-1 (Ang-1),wherein the factor is a full length protein or a functional derivativeor fragment thereof, or a nucleic acid encoding said factor orfunctional derivative or fragment thereof, or an agent that enhancesproduction and/or male erection or female sexual arousal stimulatingfunction of the factor, thereby preventing or treating male erectiledysfunction or female sexual arousal disorder in the mammal.Combinations, kits, and combinatorial methods for preventing or treatingmale erectile dysfunction or female sexual arousal disorder are alsoprovided.

BACKGROUND OF THE INVENTION

[0004] VEGF is a family of proteins that were discovered on the basis oftheir ability to stimulate VEC (vascular endothelial cell) growth(angiogenesis). It now comprises five members, namely, VEGF-A, VEGF-B,VEGF-C, VEGF-D, and PLGF (placenta growth factor) that are encoded fromdistinct genes. Achen, et al., Proc. Nat'l. Acad. Sci. USA, 95: 548(1998), Joukov, et al., EMBO J., 15: 1571 (1996), Maglione, et al.,Oncogene, 8: 925 (1993), Olofsson, et al., Proc. Nat'l. Acad. Sci. USA,93: 2576 (1996), Yamada, et al., Genomics, 42: 483 (1997). Each of thefive members in turn comprises two or more isoforms that arise by thesplicing of their respective pre-mRNAs. For example, the VEGF-A familyincludes VEGF₂₀₆, VEGF₁₈₉, VEGF₈₃, VEGF₁₆₅, VEGF₁₄₅, VEGF₁₂₁, andVEGF₁₁₁. Anthony, et al., Placenta, 15: 557 (1994), Neufeld, et al.,FASEB J., 13: 9 (1999), Lei, et al., Biochim. Biophys. Acta, 1443: 400(1998), Jingjing, et al., Ophthamol. Vis. Sci., 40: 752 (1999), Cheung,et al., Am. J. Obstet. Gynecol., 173: 753 (1995), Burchardt, et al.,Biol. Reprod., 60: 398 (1999). Among all VEGF proteins and isoforms,VEGF₁₆₅ is by far the most frequently used form of VEGF both in basicand clinical studies.

[0005] It has been shown that, among different vascular cell types(endothelial, smooth muscle cells (SMC), and fibroblasts), SMC is theprincipal source for the secreted VEGF. Pueyo, et al., Exp. Cell Res.,238: 354 (1998). Expression of VEGF in SMC is upregulated by multiplefactors including phorbol esters (Tischer, et al., J. Biol. Chem., 266:11947 (1991)), cAMP (Claffey, et al., J. Biol. Chem., 267: 16317(1992)), and hypoxia (Goldberg, et al., J. Biol. Chem., 269: 4355(1994), Shweiki, et al., Proc. Nat'l Acad. Sci. USA, 92: 768 (1995)).The secreted VEGF acts on VEC principally through two different cellsurface receptors, VEGFR-1 and VEGFR-2. Activation of VEGFR-1 results inVEC migration, while activation of VEGFR-2 VEC migration andproliferation. Waltenberger, et al., J. Biol. Chem., 269: 26988 (1994),Neufeld, et al., FASEB J., 13: 9 (1999), Ortega, et al., Front. Biosci.,4: D141 (1999). Although VEGFR-1 and VEGFR-2 have long been consideredendothelium-specific, they have both been detected in human uterine andbovine aorta SMC. Grosskreutz, et al., Microvasc. Res., 58: 128 (1999),Brown, et al., Lab. Invest., 76: 254 (1997). Cultured uterine SMCresponded to VEGF in the form of cell proliferation and cultured aortaSMC cell migration. Cultured human colon SMC, however, did not expressVEGF receptors, nor did they respond to VEGF treatment. Brown, et al.,Lab. Invest., 76: 254 (1997).

[0006] Angiogenesis is a complex process that includes activation,migration and proliferation of endothelial cells and formation of newblood vessels. D'Amore, et al., Ann. Rev. Physiol., 49(9-10): 453-64(1987). VEGF has been shown to be intimately involved in the entiresequence of events leading to growth of new blood vessels. Gross, etal., Proc. Nat'l Acad. Sci., 80(9): 2623-27 (1983), Folkman, et al.,Proc. Nat'l. Acad. Sci., 76(10): 5217-21 (1979). Five human VEGFisoforms of 121, 145, 165, 189 and 206 amino acids have been isolated.Gross, et al., Proc. Nat'l. Acad. Sci., 80(9): 2623-27 (1983), Leung, etal., Science, 246: 1306-09 (1989), Poltorak, et al., J. Biol. Chem.,272(11): 7151-78 (1997). Among the isoforms, VEGF 165 seems to be themost effective and most commonly used. The effect of VEGF 165 inaugmenting perfusion and in stimulating formation of collateral vesselshas been shown in animal models Hopkins, et al., J. Vascular Surgery,27(5): 886-94 (1998), Asahara, et al., Circulation, 91(11): 2793-801(1995), Hariawala, et al., J. Surg. Res., 63(1): 77-82 (1996), Bauters,et al., Circulation, 91(11): 2802-9 (1995), Bauters, C., et al., Am. J.Physiol., 267(4 Pt 2): H1263-71 (1994), Takeshita, et al.,. J. Clin.Invest., 93(2): 662-70 (1994), Takeshita, et al., Circulation, 90(5 Pt2): II228-34 (1994), Takeshita, et al., Am. J. Path., 147(6): 1649-60(1995), Banai, et al., Circulation, 89(5): 2183-9 (1994). In clinicaltrials, successful induction of collateral blood vessels in ischemicheart disease and critical limb ischemia by VEGF have also beenreported. Baumgartner, et al., Circulation, 97(12): 1114-23 (1998),Losordo, et al., Am. Heart J., 138(2 Pt 2): 132-41 (1999).

[0007] Platelet-derived growth factor (PDGF) is a potent mitogen forcells of mesenchymal origin, stimulating both connective tissues andneuroglial cells. PDGF also acts as a potent chemoattractant formesenchymal cells, mononuclear cells, and neutrophils. PDGF is stored inplatelet granules and released with platelet activation. Other celltypes also produce PDGF, including endothelial cells,monocytes/macrophages, vascular smooth muscle cells, fibroblasts, andcytotrophoblasts. PDGF consists of disulfide-linked dimers of αα, αβ, orββ configuration. PDGF has a short half-life and usually produces onlylocal effects. Two distinct PDGF receptors have been identified that arestructurally related and have an intracellular protein kinase domain.

[0008] Neurotrophins are a class of structurally related growth factorsthat promote neural survival and differentiation. They stimulate neuriteoutgrowth, suggesting that they can promote regeneration of injuredneurons, and act as target-derived neurotrophic factors to stimulatecollateral sprouting in target tissues that produce the neurotrophin.Korsching, J. Neurosci., 13: 2739 (1993). Recently, local synthesis andautocrine mechanisms of action have been reported. Lewin and Barde, Ann.Rev. Neurosci., 19: 289 (1996). In vivo overexpression of a neurotrophicfactor, through gene transfer, would ensure local and continuousneurotrophin production in a manner resembling the physiologic, as theseproteins are usually produced and secreted by target and glial cellssurrounding neurons.

[0009] Neurotrophin-3 (NT-3) is a member of the neurotrophin class ofstructurally related growth factors. NT-3 is a 27 kDa homodimer thatsupports the growth and survival of sympathetic neurons as well assensory neurons. NT-3 is highly conserved across species and isprimarily expressed in kidney, spleen, and heart with lower expressionlevels found in the skin, skeletal muscle, lung, thymus, and ovaries.NT-3 binds the low affinity NGF receptor, p75^(NTR), and may initiateapoptosis through this receptor. NT-3 also binds and induces signalingthrough the TrkC receptor.

[0010] Neurotrophin-4 (NT-4) is yet another member of this class ofneurotrophins. NT-4 is a homodimer that supports the growth and survivalof sympathetic neurons, dorsal root ganglion neurons, nodose ganglionneurons, basal forebrain cholinergic neurons and neurons of the locuscoeruleus. NT-4 is less highly conserved between species, unlike otherneurotrophins. NT-4 expression is widespread in brain and peripheraltissues. NT-4 induces cellular signaling through the p75^(NTR) receptoras well as the TrkB receptor.

[0011] Brain-derived neurotrophic factor (BDNF), another member of theneurotrophins, was initially characterized as a basic protein present inbrain extracts and capable of increasing the survival of dorsal rootganglia. Leibrock, et al., Nature, 341: 149 (1989). When axonalcommunication with the cell body is interrupted by injury, Schwann cellsproduce neurotrophic factors such as nerve growth factor (NGF) and BDNF.Neurotrophins are released from the Schwann cells and disperseddiffusely in gradient fashion around regenerating axons, which thenextend distally along the neurotrophins' density gradient. Ide,Neurosci. Res., 25: 101 (1996). Local application of BDNF to transectednerves in neonatal rats has been shown to prevent the massive death ofmotor neurons that follows axotomy. DiStefano, et al., Neuron, 8:983(1992), Oppenheim, et al., Nature, 360: 755 (1992), Yan, et al., Nature,360: 753 (1992). The mRNA titer of BDNF increases to several times thenormal level 4 days after axotomy and reaches its maximum at 4 weeks.Meyer, et al., J. Cell Biol., 119: 45 (1992). Moreover, BDNF has beenreported to enhance the survival of cholinergic neurons in culture.Nonomura, et al., Brain Res., 683: 129 (1995).

[0012] Angiopoietin-1 (Ang-1) is a member of a family of endotheliumgrowth factors. Ang-1 is a ligand for the Tie-2 receptor, a receptortyrosine kinase with immunoglobulin and epidermal growth factor homologydomains expressed primarily on endothelial cells and very earlyhematopoietic cells. Ang-1 promotes chemotaxis, cell survival, cellsprouting, vessel growth and stabilization of Tie-2-expressingendothelial cells. Ang-1 is thought to have a distinct angiogenic rolefrom that of VEGF involving the recruitment of peri-endothelial cellsthat will become pericytes and smooth muscle tissue of the blood vessel,thereby maintaining the stability of the blood vessels. See, e.g.,Hanahan, D., Science, 277: 48-50.

[0013] Basic fibroblast growth factor (bFGF) is a member of thefibroblast growth factor family. bFGF stimulates the proliferation ofall cells of mesodermal origin including smooth muscle cells,neuroblasts, and endothelial cells. bFGF stimulates neurondifferentiation, survival, and regeneration. In vitro functions suggestthat bFGF modulates angiogenesis, wound healing and tissue repair, andneuronal function in vivo. bFGF, a heparin-binding growth factor, iscapable of inducing functionally significant angiogenesis in models ofmyocardial and limb ischemia. Zbeng, et al., Am. J. Physiol. Heart Circ.Physiol., 280: H909-17 (2001), Laham, et al., J. Am. Coll. Cardiol., 36:2132-39 (2000), Laham, et al., Curr. Interv. Cardiol. Rep., 1: 228(1999), Unger, et al., Am. J. Cardiol., 85: 1414-19 (2000), Kawasuji, etal., Ann. Thorac. Surg., 69: 1155 (2000), Rajanayagam, et al., J. Am.Coll. Cardiol., 35: 519 (2000), Kornowski, et al., Circulation, 101:545-48 (2000), Ohara, et al., Gene Ther., 8: 837 (2001), Lazarous, etal., J. Am. Coll. Cardiol., 36: 1239 (2000), Rakue, et al., Japan Circ.J., 62: 933-39 (1998), Baffour, et al., J. Vasc. Surg., 16: 181 (1992).

[0014] Erectile function is a hemodynamic process of blood in-flow andpressure maintenance in the cavernosal spaces. Christ, Urol. Clin. NorthAm., 22: 727 (1995). Following sexual arousal and the release of nitricoxide to the erectile tissue, three processes occur to achieve anerection. These are relaxation of the trabecular smooth muscle, arterialdilation and venous compression. Id. During this final stage, arterialflow fills sinusoidal spaces, compressing subtunical venules therebyreducing venous outflow. Blood flows into the cavernous spaces of thepenis, thus expanding and stretching the penis into a rigid organ. Theflow of blood in and out of the cavernous spaces is controlled bycavernous smooth muscle cells (CSMC) embedded in the trabeculae of thecavernous spaces. With normal erectile function, a high intracavemouspressure (ICP) is maintained with a low inflow rate. Karadeniz, et al.,Urol. Int., 57: 85 (1996).

[0015] As such, the penis is a predominantly vascular organ, andvascular or penile arterial insufficiency is the most common etiology oferectile dysfunction (ED). Sinusoidal smooth muscle atrophy and collagendeposition is a common finding in men with long standing ED of variousetiologies, whether due to hormonal, neurological or vascular causes.Karadeniz, et al., Urol. Int., 57: 58 (1996). Such degradation in smoothmuscle quantity and quality leads to veno-occlusive dysfunction. Thisrepresents an end-stage muscular degeneration akin to myocardial changeswith congestive heart failure or dilated cardiomyopathy for which notreatment currently exists with hope of reversing the underlyingpathologic process.

[0016] Veno-occlusive disease is a common finding among patients witherectile dysfunction (ED). Following radical prostatectomy, for example,approximately 30% of patients may have vasculogenic ED in addition toneurogenic ED and at least half of these men may have venous leak.Regardless of the etiology of organic ED (neurogenic, traumatic,hormonal, and vascular, etc.), venous leakage is a common finalcondition resulting from smooth muscle atrophy. Mersdorf, et al., J.Urol., 154: 749 (1991). Veno-occlusive dysfunction is the most commonetiology of ED among non-responders to medical management of ED. None ofthe medical therapy currently exists is curative for this condition.Patients with veno-occlusive dysfunction exhibit a poor response tointracavernous injection with vasoactive agents (papavarine,prostaglandin El, phentolamine, or combinations, for example), despitegood arterial flow demonstrated by duplex ultrasound. The diagnosis ofveno-occlusive disease may be confirmed with specific findings oncavernosometry and cavernosography. Nehra, et al., J. Urol., 156: 1320(1996).

[0017] Atherosclerotic or traumatic arterial occlusive disease of thepudendal-cavernous-helicine arterial tree can decrease the perfusionpressure and arterial flow to the sinusoidal spaces, thus decreasing therigidity of the erect penis. Common risk factors associated withgeneralized arterial insufficiency include hypertension, hyperlipidemia,cigarette smoking, diabetes mellitus, and pelvic irradiation. Goldstein,et al., JAMA, 251: 903-910 (1984), Rosen, M. P., et al., Radiology,174(3 Pt 2): 1043-48 (1990), Levine, F. J., et al., J. Urology, 144(5):1147-53 (1990). Epidemiological studies have shown a high incidence ofED in patients with coronary arterial disease. Heaton, J. P., et al.,Int'l J. Impotence Res., 8(1): 35-39 (1996). Focal lesion of the commonpenile or cavernous artery is most often seen in young patients who havesustained blunt pelvic or perineal trauma such as in cases of bikingaccidents. Levine, F. J., et al., J. Urology, 144(5): 1147-53 (1990).

[0018] Because of the close proximity of the cavernous nerves to thecapsule of the prostate, ED is a frequent complication after radicalprostatectomy or cystectomy and prostatic cryosurgery. Although thenerve-sparing prostatectomy technique developed by Walsh, et al., Br. J.Urol., 56: 694 (1984) has significantly reduced the postoperativeimpotence rate, a large number of patients still suffer from inadequatepenile rigidity. Peripheral nerve regeneration is a slow process, andthe fact that most patients do not recover potency for 6 months to 2years indicates substantial axonal damage, even with preservation of theneural sheath. An anatomic study of the cavernous nerves by Paick etal., Urology, 42: 145 (1993) revealed both a medial and a lateral bundleof cavernous nerves at the level of the prostate, suggesting that insome cases the lateral bundle can be saved, even in non-nerve-sparingprostatectomy.

[0019] The sprouting of the remaining nerves in penile tissue appears tobe more important in regeneration than re-growth of nerves through thedamaged and fibrotic tissues. The importance of sprouting in theremaining nerves was confirmed in an animal study that revealedregeneration of the cavernous nerves after unilateral resection.Carrier, et al., J. Urol., 153: 1722 (1995). In addition, a previousstudy in our laboratory showed that systemic growth hormone injectionsignificantly enhanced cavernous nerve regeneration after unilateralinjury. Jung, et al., J. Urol., 160: 1899 (1998).

[0020] Methods for treating erectile dysfunction have included from theadministration of prostaglandin E (U.S. Pat. No. 5,942,545), localadministration of vascular muscle relaxants and vasoactivepharmaceutical agents. See, for example, U.S. Pat. Nos. 5,942,545,6,056,966; and 5,646,181.

[0021] Advancement in molecular biology has brought improvedunderstanding of pathophysiology on the gene and molecular level, andoffers promise of treatment possibilities aimed at a specific pathologicmolecular mechanism. As in other vasculopathies such as limbclaudication (Baumgartner, et al., Circulation, 97: 1114 (1998) andcoronary artery disease (Symes, et al., Ann. Thorac. Surg., 68: 830(1999), treatment with VEGF in either protein or gene form has increasedneovascularity in animal models and improved symptomatic angina andwound healing in humans with inoperable heart disease and critical limbischemia, respectively. The penis represents a convenient tissue targetfor gene or growth factor therapy due to the penis' external location onthe body, ubiquity of endothelial-lined spaces and low-level blood flowin the flaccid state. In addition, the penis is filled with billions ofendothelial and smooth muscle cells both are rich in VEGF receptors.Liu, et al., J. Urol., 166: 354-360 (2001).

[0022] Recently, we have established an animal model in which CSMC wasseen decreased following internal iliac artery ligation that restrictedblood supply to the penis. However, rats treated with intracavernousinjection of vascular endothelial growth factor (VEGF) shortly afterinternal iliac artery ligation had nearly normal CSMC. The protectiveeffects of VEGF on CSMC could be due to partial restoration of bloodsupply as VEGF is expected to stimulate vascular endothelial cell (VEC)proliferation. Lin, et al., Proc. Nat'l Acad. Sci. USA, 97: 10242-47(2000). Alternatively, VEGF might act directly on CSMC, as we willpresent evidence that CSMC express one of the two principal VEGFreceptors. Sondell, et al., Eur. J. Neurosci., 12: 4243-54 (2000); Liu,et al., J. Urol., 166: 354-360 (2001).

[0023] Females can also have sexual dysfunction, and this dysfunctioncan increase with age. It is usually associated with the presence ofvascular risk factors, genital smooth muscle atrophy, and onset ofmenopause. Some of the vascular and muscular mechanisms that contributeto penile erection in the male are believed to be similar vasculogenicfactors in the female genital response. It is known that in women sexualarousal is accompanied by arterial inflow which engorges the vagina andincreases vaginal lubrication, and that the muscles in the clitoris andthe perineum assist in achieving clitoral erection.

[0024] In the female patient, sexual arousal disorder can arise fromorganic and pyschogenic causes, or from a combination of the foregoing.Female sexual arousal disorder is classified into five categories: 1)hypoactive sexual desire disorder, 2) sexual aversion disorder, 3)sexual arousal disorder, 4) orgasmic disorder, and 5) sexual paindisorder. The present invention applies to sexual arousal disorder.Sexual arousal disorder is the persistent or recurring inability toattain or maintain adequate sexual excitement, causing personaldistress. It may be experienced as the lack of subjective excitement orthe lack of genital lubrication or swelling or other somatic responses.Organic female sexual arousal disorder is known to be related in part tovasculogenic impairment resulting in inadequate blood flow, vaginalengorgement insufficiency and clitorial erection insufficiency. Animalstudies have demonstrated the dependence of vaginal vascular engorgementand clitoral erection on blood flow. See, for example, Park et al.,“Vasculogenic female sexual dysfunction: the hemodynamic basis forvaginal engorgement insufficiency and clitoral erectile insufficiency,”Int'l J. Impotence Res., 9(1), 27-37 (March 1997).

[0025] Female sexual dysfunction has been treated with pharmacologicalintervention to stimulate blood flow as well as with prostaglandins.See, for example, U.S. Pat. Nos. 6,193,992 B1; 5,945,117; 6,031,002; and5,891,915.

SUMMARY OF THE INVENTION

[0026] The present invention provides for methods, combinations, andkits for the treatment and prevention of male erectile dysfunction orfemale sexual arousal disorder. These methods, combinations, and kitsinvolve the administration of vascular endothelial growth factor (VEGF),brain-derived growth factor (BDNF), basic fibroblast growth factor(bFGF), neurotrophin-3 (NT-3), neurotrophin-4 (NT-4), platelet-derivedgrowth factor (PDGF), angiopoietin-1 (Ang-1), or a combination thereof.

[0027] In one aspect, the present invention provides for a method forpreventing or treating male erectile dysfunction or female sexualarousal disorder, which method comprises administering to a mammal towhom such prevention or treatment is needed or desirable, an effectiveamount of a factor, wherein the factor is VEGF, BDNF, bFGF, NT-3, NT-4,PDGF, Ang-1, an agent that enhances production and/or male erection orfemale sexual arousal stimulating function of the factor, or acombination thereof, thereby preventing or treating the male erectiledysfunction or the female sexual arousal disorder in the mammal.Preferably, the factor is a full length protein or a functionalderivative or fragment thereof or a nucleic acid encoding the factor orfunctional derivative or fragment thereof.

[0028] In a specific embodiment, the mammal is a human and the factor,or a functional derivative or fragment thereof, or the nucleic acidencoding the factor, or a functional derivative or fragment thereof, isof human origin.

[0029] Preferably, the factor protein or nucleic acid, or a functionalderivative or fragment thereof, is administered by intracavernousinjection, subcutaneous injection, intravenous injection, intramuscularinjection, intradermal injection, or topical administration.

[0030] In a specific embodiment, the factor nucleic acid, or afunctional derivative or fragment thereof, is administered via a genetherapy vector, preferably the gene therapy vector is an adenovirusassociated vector, a retroviral vector, an adenovirus vector, or alentivirus vector. More preferably, the gene therapy vector is anadenovirus associated vector.

[0031] In yet another specific embodiment, the factor protein, or afunctional derivative or fragment thereof, is administered via aliposome.

[0032] In another specific embodiment, the factor nucleic acid, or afunctional derivative or fragment thereof, is administered via aliposome.

[0033] Preferably, the male erectile dysfunction to be treated orprevented is erectile dysfunction induced by or secondary to nervedysfunction, arterial insufficiency, venous leakage, severe vascularinsufficiency, mild vascular disease, hormonal insufficiency, drug use,surgery, chemotherapy, or radiation.

[0034] Preferably, the female sexual arousal disorder to be treated orprevented is sexual dysfunction induced by or secondary to nervedysfunction, arterial insufficiency, severe vascular insufficiency, mildvascular disease, hormonal insufficiency, drug use, surgery,chemotherapy, or radiation.

[0035] In a specific embodiment, the factor protein or a functionalderivative or fragment thereof, or a nucleic acid encoding the factor orfunctional derivative or fragment thereof, or an agent that enhancesproduction and/or female sexual arousal stimulating function of thefactor, is administered in an amount sufficient to improve blood flowand regenerate nerve and smooth muscle in the clitoris and vaginal wall.

[0036] In another specific embodiment, the factor protein or afunctional derivative or fragment thereof, or a nucleic acid encodingthe factor or functional derivative or fragment thereof, or an agentthat enhances production and/or female sexual arousal stimulatingfunction of the factor, is administered in a cream or via injection tothe clitoris and vaginal wall of the patient.

[0037] In yet another specific embodiment, the factor protein or afunctional derivative or fragment thereof, or a nucleic acid encodingthe factor or functional derivative or fragment thereof, or an agentthat enhances production and/or the male erection or female sexualarousal stimulating function of the factor, is administered byintracavemous injection.

[0038] In a preferred embodiment, the male erectile dysfunction orfemale sexual arousal dysfunction is induced by or secondary to nerveinjury, and the combination of factors administered are: a) VEGF andNT-3, b) VEGF and NT-4, or c) VEGF and BDNF.

[0039] In another preferred embodiment, the male erectile dysfunction orfemale sexual arousal disorder is induced by or secondary to severevascular insufficiency and the combination of factors administered are:a) VEGF and PDGF, b) VEGF and bFGF, or c) VEGF and Ang-1.

[0040] In yet another preferred embodiment, the male erectiledysfunction or female sexual arousal dysfunction is induced by orsecondary to mild vascular disease and the factor administered is VEGF.

[0041] In another aspect, the present invention provides for acombination for preventing or treating male erectile dysfunction orfemale sexual arousal disorder, which combination comprises: a) aneffective amount of an agent that stimulates male erectile or femalesexual function; and b) an effective amount of a factor, wherein thefactor is a full length protein or a functional derivative or fragmentthereof, or a nucleic acid encoding said factor or functional derivativeor fragment thereof, an agent that enhances production and/or maleerection or female sexual arousal stimulating function of said factor,or a combination thereof, and the factors include VEGF, BDNF, bFGF,NT-3, NT-4, PDGF, and Ang-1.

[0042] In yet another aspect, the present invention provides acombination for preventing or treating male erectile dysfunction orfemale sexual arousal disorder induced by or secondary to nerve injury,which combination comprises: a) an effective amount of VEGF, wherein theVEGF is a full length protein or a functional derivative or fragmentthereof, or a nucleic acid encoding the VEGF or functional derivative orfragment thereof; and b) an effective amount of a factor selected fromthe group consisting of NT-3, NT-4, and BDNF, wherein the factor is afull length protein or a functional derivative or fragment thereof, or anucleic acid encoding the factor or functional derivative or fragmentthereof, or an agent that enhances production and/or male erection orfemale sexual arousal stimulating function of the factor.

[0043] In another aspect, the present invention provides a combinationfor preventing or treating male erectile dysfunction or female sexualarousal disorder induced by or secondary to severe vascularinsufficiency, which combination comprises: a) an effective amount ofVEGF, wherein the VEGF is a full length protein or a functionalderivative or fragment thereof, or a nucleic acid encoding the VEGF orfunctional derivative or fragment thereof; and b) an effective amount ofa factor selected from the group consisting of PDGF, bFGF, and Ang-1,wherein the factor is a full length protein or a functional derivativeor fragment thereof, or a nucleic acid encoding the factor or functionalderivative or fragment thereof, or an agent that enhances productionand/or male erection or female sexual arousal stimulating function ofthe factor.

[0044] In yet another aspect, the present invention provides for theabove-described combinations, preferably in the form of a pharmaceuticalcomposition, that can be used for preventing or treating male erectiledysfunction or female sexual arousal disorder. The above-describedcombinations can further comprise a pharmaceutically acceptable carrieror excipient.

[0045] In another aspect, the present invention provides a method forpreventing or treating male erectile dysfunction or female sexualarousal disorder, which method comprises administering an effectiveamount of the above described combinations to a mammal in need thereof,thereby preventing or treating the male erectile dysfunction or thefemale sexual arousal disorder in the mammal.

[0046] In one aspect, the present invention provides for a kitcomprising the above-described combinations and an instruction for usingthe combination in treating or preventing male erectile dysfunction orfemale sexual arousal disorder.

[0047] In yet another aspect, the present invention provides a method ofpromoting sprouting of new nerve fibers from blood vessel explants,which method comprises the steps of: a) isolating a blood vessel; b)attaching the blood vessel to a media-coated coverslip; and c)incubating with a growth-stimulating compound. In one preferredembodiment, the growth-stimulating compound is VEGF. The presentinvention also provides for a method of identifying a compound forpromoting sprouting of new nerve fibers from blood vessel explants,which method comprises assaying candidate compounds for nerve growthpromoting activity ex vivo using the above described method andidentifying a compound that promotes nerve growth in a blood vesselexplant as indicative of a compound that promotes nerve growth.

[0048] In another aspect, the present invention provides for a method ofinducing angiogenesis, which method comprises the steps of: a)co-culturing an isolated blood vessel and an isolated muscle explant;and b) incubating with a growth-stimulating compound. In a preferredembodiment, the compound is a combination of VEGF and PDGF. The presentinvention also provides for a method of identifying a compound forinducing angiogenesis, which method comprises assaying candidatecompounds for angiogenic activity ex vivo using the method describedabove, and identifying a compound that promotes angiogenesis in a bloodvessel cell as indicative of a compound that promotes angiogenesis.

[0049] In yet another aspect, the present invention provides for amethod for promoting growth of cavernous nerves from major pelvicganglia (MPG), which method comprises contacting the MPG with aneffective amount of a factor, wherein the factor is selected from thegroup consisting of vascular endothelial growth factor (VEGF),brain-derived growth factor (BDGF), basic fibroblast growth factor(bFGF), neurotrophin-3 (NT-3), neurotrophin-4 (NT-4), platelet-derivedgrowth factor (PDGF), and angiopoietin-1 (Ang-1), thereby promotinggrowth of the cavernous nerves from the MPG.

[0050] In another aspect, the present invention provides for a method ofidentifying a compound that promotes growth of cavernous nerves frommajor pelvic ganglia (MPG), which method comprises: a) in vitroculturing MPG; b) measuring growth of cavernous nerves from the MPG inthe presence and absence of a candidate compound; and c) identifying acompound that promotes nerve growth as indicative of a compound thatpromotes growth of cavernous nerves from MPG.

[0051] Other aspects of the invention are described throughout thespecification.

BRIEF DESCRIPTION OF THE DRAWINGS

[0052]FIG. 1. Electrostimulation of the cavernous nerve at 8 weeks: A)sham operation group; B) LacZ group; and C) BDNF group. Note highermaximal intracavernous pressure in the BDNF than in the LacZ group. Scanrate=10/sec.

[0053]FIG. 2. Quantification of VEGF secreted by CSMC. Equal number(4×10⁵) of CSMC from different-aged rats were seeded in each well of6-well plates and allowed to grow for three days in medium containing10% of FBS. The medium from each well was then assayed for theconcentration of rat-specific VEGF (panel B) and the cell number wasdetermined (panel A). The calculated amount of VEGF in each well wasthen divided by the number of cells in each well to derive the datashown in panel C.

[0054]FIG. 3. Effects of VEGF on the growth rates of VSMC and CSMC.Equal numbers (5,000) of VSMC (from 16-weeks-old rats, panel A) and CSMC(from rats of indicated ages, panels B to I) were seeded in each well of96-well plates and allowed to grow for three days in media containingthe indicated concentrations of VEGF. The final numbers of cells weredetermined with a proliferation assay kit and expressed as opticaldensity (OD_(490nm)) values. These values were converted into % ofcontrol (shown on the left of panels A through J) with the value of thegrowth rate in medium containing no added VEGF being referred to ascontrol (i.e., 100%). Panel J compares the growth rates of CSMC fromrats of the indicated ages in media containing the optimal concentration(12.5 ng/ml) of VEGF.

[0055]FIG. 4. Effects of VEGF on the mobility of VSMC and CSMC. Equalnumbers (8,000) of VSMC (from 16-weeks-old rats, panel A) and CSMC (fromrats of indicated ages, panels B to I) were loaded in each well of24-well Transwell plates and allowed to migrate through a membranetoward a medium containing the indicated concentration of VEGF. Fourhours later, the numbers of cells that had migrated through the membranewere counted under a microscope. These numbers are indicated on the leftof the panels. Panel J compares the numbers of CSMC (from rats of theindicated ages) that had migrated through the membrane toward mediacontaining the optimal concentration (10 ng/ml) of VEGF.

[0056]FIG. 5. Identification of VEGFR-1 and VEGFR-2 mRNA expression.RNAs of the following cells and tissues (lanes 1-15) were subjected toRT-PCR with primer pair VEGFR-1s and VEGFR-1a, primer pair VEGFR-2s andVEGFR-2a, and primer pair β-actin-s and β-actin-a (Table 1). Thereaction products were electrophoresed in a 1.5% agarose gel and stainedwith ethidium bromide. The RT-PCR product in each lane was derived from25 ng (for VEGFR-1 and VEGFR-2) or 1 ng (for β-actin) of total cellularor tissue RNAs. M, 100-bp size marker. Lane 1, CSMC from 1-week-oldrats; lane 2, CSMC from 2-weeks-old rats; lane 3, CSMC from 3-weeks-oldrats; lane 4, CSMC from 4-weeks-old rats; lane 5, CSMC from 6-weeks-oldrats; lane 6, CSMC from 11-weeks-old rats; lane 7, CSMC from16-weeks-old rats; lane 8, CSMC from 28-months-old rats; lane 9, aortaSMC from 16-weeks-old rats; lane 10, heart of a 16-weeks-old rat; lane11, aorta of a 16-weeks-old rat; lane 12, corpus cavemosum of a16-weeks-old rat.

[0057]FIG. 6. Identification of VEGFR-1 protein expression. Proteinextracts of CSMC from rats of the following ages (lanes 1-8) wereelectrophoresed in 7.5% SDS-PAGE and then transferred to PVDF membrane.Detection of VEGFR-1 protein on the membrane was performed by the ECLprocedure using an anti-VEGFR-1 rabbit serum. Lane 1, 1-week-old; lane2, 2-weeks-old; lane 3, 3-weeks-old; lane 4, 4-weeks-old; lane 5,6-weeks-old; lane 6, 11-weeks-old; lane 7, 16-weeks-old; lane 8,28-months-old.

DETAILED DESCRIPTION OF INVENTION

[0058] A. Definitions

[0059] Unless defined otherwise, all technical and scientific terms usedherein have the same meaning as is commonly understood by one ofordinary skill in the art to which this invention belongs. All patents,patent applications and other publications and sequences from GenBankand other databases referred to herein are incorporated by reference intheir entirety. If a definition set forth in this section is contrary toor otherwise inconsistent with a definition set forth in patents, patentapplications and other publications and sequences from GenBank and otherdatabases that are herein incorporated by reference, the definition setforth in this section prevails over the definition that is incorporatedherein by reference.

[0060] As used herein, “a” or “an” means “at least one” or “one ormore.”

[0061] As used herein, an “erectile dysfunction (or impotence)” refersto the inability of a male mammal, e.g., a man, to achieve and maintainpenile erection for satisfactory sexual intercourse.

[0062] As used herein, a “female sexual arousal disorder” refers to thepersistent or recurring inability to attain or maintain adequate sexualexcitement causing personal distress. It may be experienced as lack ofsubjective excitement or lack of genital (lubrication and swelling) orother somatic responses.

[0063] As used herein, “vascular endothelial growth factor (VEGF)” or“brain-derived neurotrophic factor (BDNF)” or “basic fibroblast growthfactor (bFGF)” or “platelet-derived growth factor (PDGF)” or“neurotrophin-3 (NT-3)” or “neurotrophin-4 (NT-4)” or “angiopoietin-1(Ang-1)” includes those variants with conservative amino acidsubstitutions that do not substantially alter their male erection orfemale sexual arousal-stimulating activity. Suitable conservativesubstitutions of amino acids are known to those of skill in this art andmay be made generally without altering the biological activity of theresulting molecule. Those of skill in this art recognize that, ingeneral, single amino acid substitutions in non-essential regions of apolypeptide do not substantially alter biological activity (see, e.g.,Watson et al. Molecular Biology of the Gene, 4th Edition, 1987, TheBejacmin/Cummings Pub. co., p.224).

[0064] As used herein, a “functional derivative or fragment of a factor”refers to a derivative or fragment of the factor that stillsubstantially retains its function as an erection or sexual arousalstimulant. Normally, the derivative or fragment retains at least 50% ofits erection or sexual function stimulating activity. Preferably, thederivative or fragment retains at least 60%, 70%, 80%, 90%, 95%, 99% and100% of its erection or sexual function stimulating activity.

[0065] As used herein, an “agent that enhances production of the factor”refers to a substance that increases transcription and/or translation ofa factor gene, or a substance that increases post-translationalmodification and/or cellular trafficking of a factor precursor, or asubstance that prolongs half-life of a factor protein.

[0066] As used herein, an “agent that stimulates erection or femalesexual arousal function activity of a factor” refers to a substance thatincreases potency of the factor's erection or sexual arousal stimulatingactivity, or a substance that increases sensitivity of a factor'snatural ligand in an erection or sexual arousal stimulation signalingpathway, or a substance that decreases potency of a factor's antagonist.Such an agent is not VEGF, BDNF, bFGF, PDGF, NT-3, NT-4, or Ang-1.

[0067] As used herein, a “combination” refers to any association betweentwo or among more items.

[0068] As used herein, a “composition” refers to any mixture of two ormore products or compounds. It may be a solution, a suspension, liquid,powder, a paste, aqueous, non-aqueous or any combination thereof.

[0069] As used herein, a “nerve dysfunction” refers to the inability ofthe penis to hold the blood during erection or the persistent orrecurring inability to attain or maintain adequate sexual excitement ina male or female mammal causing personal distress including, but notlimited to dysfunction caused by diabetes mellitus, hypertension,hyperlipidemia, penile injury, aging, pelvic surgery or irradiation.

[0070] As used herein, an “arterial insufficiency” refers to reducedperfusion pressure and arterial flow associated with trauma or diseaseincluding, but not limited to, that associated with hypertension,hyperlipidemia, cigarette smoking, diabetes mellitus, and pelvicirradiation.

[0071] As used herein, a “venous leakage” refers to the inability of thepenis to hold the blood during erection caused by a disorder including,but not limited to diabetes mellitus, hypertension, hyperlipidemia,penile injury, aging, pelvic surgery or irradiation.

[0072] As used herein, a “hormonal insufficiency” refers to a groupcomprising, but not limited to, perimenopausal-, post menopausal-,cancer-related-, hypogonadism-, and osteoporosis-hormonalinsufficiencies.

[0073] As used herein, a “drug use” includes pharmaceutical drug use andsubstance abuse.

[0074] As used herein, a “surgery” refers to the performance of anoperation including reconstructive, cosmetic, and restorative proceduresand removal of an organ or tissue or some portion thereof.

[0075] As used herein, a “radiation” refers to treatment by photons,electrons, neutrons or other ionizing radiation.

[0076] As used herein, a “chemotherapy” refers to the administration ofany agent that mediates the regression of malignant growth by inducingcell death or retarding cell growth through DNA damaging and non-DNAdamaging mechanisms.

[0077] As used herein, a “nerve injury” refers to damage of the somaticnerve or autonamic nerve that controls sensation and blood flow to thepenis, vagina or the clitoris. The injury can occur as a result of bluntor penetrating trauma to the pelvis, perineum or the penis as well assurgery or irradiation of the pelvic organs or the external genitalia.

[0078] As used herein, a “severe vascular insufficiency” refers to nearcomplete cessation of blood flow to the penis, vagina, or clitoris dueto damage of both internal pudendal or penile arteries.

[0079] As used herein, a “mild vascular insufficiency” refers to mild tomoderate restriction of blood flow to the penis, vagina, or clitoris andthus impairs erectile function or female sexual arousal due to partialdamage to the pudenal or penile arteries.

[0080] For clarity of disclosure, and not by way of limitation, thedetailed description of the invention is divided into the subsectionsthat follow.

[0081] B. Methods to Prevent or Treat Male Erectile Dysfunction orFemale Sexual Arousal Disorder

[0082] In one aspect, the present invention provides for a method forpreventing or treating male erectile dysfunction or female sexualarousal disorder, which method comprises administering to a mammal towhom such prevention or treatment is needed or desirable, an effectiveamount of a factor, wherein the factor is VEGF, BDNF, bFGF, NT-3, NT-4,PDGF, or Ang-1, thereby preventing or treating the male erectiledysfunction or the female sexual arousal disorder in the mammal.Preferably, the factor is a full length protein or a functionalderivative or fragment thereof, or a nucleic acid encoding the factor orfunctional derivative or fragment thereof, or an agent that enhancesproduction and/or male erection or female sexual arousal stimulatingfunction of the factor.

[0083] Any mammal can be treated with the present method. Preferably,male erectile dysfunction or female sexual arousal disorder in humansare treated or prevented. When human patients are treated, any suitablefactor protein, or a functional derivative or fragment thereof, or anysuitable factor nucleic acid, or a functional derivative or fragmentthereof, can be used. Preferably, the factor protein, or a functionalderivative or fragment thereof, or factor nucleic acid, or a functionalderivative or fragment thereof, is of human origin. But, suitable factorprotein, or a functional derivative or fragment thereof, or factornucleic acid, or a functional derivative or fragment thereof, ofnon-human origin can also be used when the non-human factor binds andstimulates the cell through the human factor receptor throughcross-reactivity.

[0084] VEGF, BDNF, bFGF, PDGF, NT-3, NT-4, or Ang-1 proteins orfunctional derivatives or fragments thereof, or a nucleic acid encodingVEGF, BDNF, bFGF, PDGF, NT-3, NT-4, Ang-1, or functional derivatives orfragments thereof, can be prepared by any methods known in the art,e.g., synthetic methods, recombinant methods or a combination thereof.Any suitable DNA construct encoding VEGF, BDNF, bFGF, PDGF, NT-3, NT-4,or Ang-1 can be used in the present invention. Such constructs include,but are not limited to VEGF-GenBank accession number M32977 (SEQ IDNO:9), BDNF-GenBank accession number M61176 (SEQ ID NO:10), bFGF-GenBankaccession number E02544 (SEQ ID NO:11), NT-3-GenBank accession numberM37763 (SEQ ID NO: 12), NT-4-GenBank accession number M86528 (SEQ ID NO:13), and PDGF-GenBank accession number X02811 (SEQ ID NO:14) (PDGF-α)and X03795 (SEQ ID NO:15) (PDGF-β), and Ang-1-GenBank accession numberU83508 (SEQ ID NO:16). Further contemplated for use in the presentinvention are the DNA sequences and resultant proteins described in U.S.Pat. Nos. 5,607,918, 5,438,121, 5,229,500, 5,180,820, 5,387,673,5,155,214, 5,026,839, and 6,037,320.

[0085] Any suitable method for the production or the stabilization ofthe VEGF, BDNF, bFGF, PDGF, NT-3, NT-4, or Ang-1 protein known to askilled artisan may be used in this invention. Formulations that may beused in these inventions include, but are not limited to those describedin U.S. Pat. Nos. 5,217,954, 5,235,043, 5,986,070, 6,077,829, 5,130,418,5,188,943, and 5,770,228.

[0086] The formulation, dosage and route of administration of theabove-described compositions, combinations, preferably in the form ofpharmaceutical compositions, can be determined according to the methodsknown in the art (see e.g., Remington: The Science and Practice ofPharmacy, Alfonso R. Gennaro (Editor) Mack Publishing Company, April1997; Therapeutic Peptides and Proteins: Formulation, Processing, andDelivery Systems, Banga, 1999; and Pharmaceutical FormulationDevelopment of Peptides and Proteins, Hovgaard and Frkjr (Ed.), Taylor &Francis, Inc., 2000; Medical Applications of Liposomes, Lasic andPapahadjopoulos (Ed.), Elsevier Science, 1998; Textbook of Gene Therapy,Jain, Hogrefe & Huber Publishers, 1998; Adenoviruses: Basic Biology toGene Therapy, Vol. 15, Seth, Landes Bioscience, 1999; BiopharmaceuticalDrug Design and Development, Wu-Pong and Rojanasakul (Ed.), HumanaPress, 1999; Therapeutic Angiogenesis: From Basic Science to the Clinic,Vol. 28, Dole et al. (Ed.), Springer-Verlag New York, 1999). Thecompositions, combinations or pharmaceutical compositions can beformulated for oral, rectal, topical, inhalational, buccal (e.g.,sublingual), parenteral (e.g., subcutaneous, intramuscular, intradermal,intracavernous, or intravenous), transdermal administration or any othersuitable route of administration. The most suitable route in any givencase will depend on the nature and severity of the condition beingtreated and on the nature of the particular composition, combination orpharmaceutical composition which is being used.

[0087] Any form of male erectile or female sexual arousal disorder canbe treated by the present method. The present invention provides amethod for the treatment or prevention of male erectile dysfunctioninduced by or secondary to nerve dysfunction, nerve injury, arterialinsufficiency, venous leakage, severe vascular insufficiency, mildvascular disease, hormonal insufficiency, drug use, surgery,chemotherapy or radiation. In another aspect, the present inventionprovides a method for the treatment and prevention of female sexualarousal disorder induced by or secondary to nerve dysfunction, nerveinjury, arterial insufficiency, severe vascular insufficiency, mildvascular disease, hormonal insufficiency, drug use, surgery,chemotherapy or radiation.

[0088] In a preferred embodiment, the factor protein or a functionalderivative or fragment thereof, or a nucleic acid encoding the factor orfunctional derivative or fragment thereof, or an agent that enhancesproduction and/or female sexual arousal stimulating function of thefactor, is administered in an amount sufficient to improve blood flowand regenerate nerve and smooth muscle in the clitoris and vaginal wall.A preferred route of administration is topically in a cream or viainjection to the clitoris and vaginal wall of the patient.

[0089] In another preferred embodiment, the present invention providesfor a method of treatment for the treatment of male erectile dysfunctionor female sexual arousal disorder by administering the factor protein ora functional derivative or fragment thereof, or a nucleic acid encodingthe factor or functional derivative or fragment thereof, or an agentthat enhances production and/or the male erection or female sexualarousal stimulating function of the factor, by intracavemous injection.

[0090] C. Combinations and Kits

[0091] In another aspect, the present invention provides for acombination for preventing or treating male erectile dysfunction orfemale sexual arousal disorder, which combination comprises: a) aneffective amount of an agent that stimulates male erectile or femalesexual function; and b) an effective amount of one or more factors,wherein the factor is a full length protein or a functional derivativeor fragment thereof, or a nucleic acid encoding said factor orfunctional derivative or fragment thereof, or an agent that enhancesproduction and/or male erection or female sexual arousal stimulatingfunction of said factor and said factors are selected from the groupconsisting of vascular endothelial growth factor (VEGF), brain-derivedgrowth factor (BDNF), basic fibroblast growth factor (bFGF),neurotrophin-3 (NT-3), neurotrophin-4 (NT-4), platelet-derived growthfactor (PDGF), and angiopoietin-1 (Ang-1).

[0092] The instant invention may further comprise the coadministrationof an agent that enhances, complements, or is synergistic with theerectile stimulating or female sexual arousal stimulating activity ofVEGF, BDNF, bFGF, PDGF, NT-3, NT-4, or Ang-1, or some combinationthereof. The agent may comprise an agent with independent pharmacologicactivity or one that prolongs the functional or structural half-life ofVEGF, BDNF, bFGF, PDGF, NT-3, NT-4, or Ang-1, or some combinationthereof. This invention also contemplates the administration of suchVEGF, BDNF, bFGF, PDGF, NT-3, NT-4, or Ang-1, a combination thereof,with or without an accompanying agent simultaneously or separately tomaximize the male erectile or female sexual arousal stimulatingactivity.

[0093] The present invention provides for administration of acombination of factors to treat or prevent male erectile dysfunction orfemale sexual arousal disorder. The combinations of VEGF and NT-3, VEGFand NT-4, or VEGF and BDNF are contemplated as useful combinations whenthe dysfunction or disorder is induced by or secondary to nerve injury.The combinations of VEGF and PDGF, VEGF and bFGF, and VEGF and Ang-1 arecontemplated as useful combinations when the dysfunction or disorder isinduced by or secondary to severe vascular insufficiency. Theadministration of VEGF alone is contemplated as useful when thedysfunction or disorder is induced by or secondary to mild vasculardisease.

[0094] In a further embodiment of the invention, the VEGF, BDNF, bFGF,PDGF, NT-3, NT-4, or Ang-1, or some combination thereof may be deliveredto said mammal using a gene therapy vector. The exemplary vectorsinclude, but are not limited to retroviruses, adenoviruses,adeno-associated viruses, lentiviruses, herpesviruses, and vacciniaviruses vectors. Replication-defective recombinant adenoviral vectorscan be produced in accordance with known techniques. See, Quantin, etal., Proc. Natl. Acad. Sci. USA, 89:2581-2584 (1992);Stratford-Perricadet, et al., J. Clin. Invest., 90:626-630 (1992); andRosenfeld, et al., Cell, 68:143-155 (1992).

[0095] The vector may include an expression construct of VEGF, BDNF,bFGF, PDGF, NT-3, NT-4, or Ang-1 nucleic acid, a functional derivativefragment thereof under the transcription control of a promoter. In apreferred embodiment, the gene therapy vector may be administered byintracavemous injection of about 0.5 to 2 ml of AAV-VEGF, AAV-BDNF,AAV-bFGF, AAV-PDGF, AAV-NT-3, AAV-NT-4, or AAV-Ang-1 at a concentrationof about 10¹⁰ virus titer. An example of a suitable promoter that may beused is the 763-base-pair cytomegalovirus (CMV) promoter. Viralpromoters, cellular promoters/enhancers and induciblepromoters/enhancers that could be used in combination with the nucleicacid contemplated for use in this invention include those listed in Jin,et al., U.S. Pat. No. 6,251,871. The expression construct may beinserted into a vector, such as pUC118, pBR322, or other known plasmidvectors, that includes an origin of replication. See, for example,Current Protocols in Molecular Biology, Ausubel, et al. eds., John Wiley& Sons, Inc. (2000), Sambrook, et al., Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Laboratory press, (1989). The plasmid vectormay also include a selectable marker such as the β-lactamase gene forampicillin resistance, provided that the marker polypeptide does notadversely effect the metabolism of the organism being treated.

[0096] The preferred embodiment of the VEGF, BDNF, bFGF, PDGF, NT-3,NT-4, or Ang-1, or agent nucleic acid may be delivered via anadeno-associated viral (AAV) vector. These viruses are single-strandedDNA, nonautotomous parvoviruses that are able to integrate efficientlyinto the genome of nondividing cells of a very broad host range.Although ubiquitous in nature, AAV has not been shown to be associatedwith any known human disease and does not elicit an immune response inan infected human host. GOODMAN & GILMAN, PHARMACOLOGICAL BASIS OFTHERAPEUTICS, 9th ed., McGraw-Hill Press (1996), p.77-101. The method toproduce purified replication deficient recombinant adeno-associatedvirions is described in Dwarki, et al., U.S. Pat. No. 6,221,646 B1, andits contents are incorporated in their entirety herein.

[0097] The present invention contemplates the use of AAV vectors thatare known to those of skill in the art. For example, the vectors andvector production methods described in U.S. Pat. Nos. 5,589,377;5,753,500; and 5,693,531. In another embodiment of this invention, thenucleic acid may be delivered in a retroviral vector, using vector andproduction method known in the art. See, for example, U.S. Pat. No.5,830,725. A further embodiment would deliver the nucleic acid in anadenovirus vector, using vectors and vector production methods known inthe art. See, for example, U.S. Pat. Nos. 6,063,622; 6,083,750;5,994,128; and 5,981,225.

[0098] In a further embodiment of the invention, factors used in thepresent method and the encoding nucleic acids may be delivered in aliposome. Liposomes are vesicular structures characterized by aphospholipid bilayer membrane and an inner aqueous medium. Multilamellarliposomes have multiple lipid layers separated by aqueous medium. Theyform spontaneously when phospholipids are suspended in an excess ofaqueous solution. The lipid components undergo self-rearrangement beforethe formation of closed structures and entrap water and dissolvedsolutes between the lipid bilayers (Ghosh and Bachhawat, 1991). For areview of the procedures for liposome preparation, targeting anddelivery of contents, see Mannino and Gould-Fogerite, Bio Techniques,6:682 (1988). See also, Current Protocols in Molecular Biology, Ausubel,et al. eds., John Wiley & Sons Press (2000), Chapters 9 and 16. Theprocess of making and loading the liposomes with nucleic acid or proteinmay employ techniques known in the art. See, for example, U.S. PatentNos., 6,007,838; 6,197,333 B1; 6,133,026; 6,120,798; 5,939,096;5,662,931; 5,552,157; and 5,270,053.

[0099] According to the present invention, the VEGF, BDNF, bFGF, PDGF,NT-3, NT-4, or Ang-1 peptides, proteins, polynucleotides, nucleic acids,or agent that enhances production and/or erection or sexual arousalstimulating function of said factor may be formulated for intracavemousinjection, subcutaneous injection, intravenous injection, intramuscularinjection, intradermal injection, or topical administration. The methodmay employ formulations for injectable administration in unit dosageform, in ampules or in multidose containers, with an added preservative.The formulations may take such forms as suspensions, solutions oremulsions in oily or aqueous vehicles, and may contain formulatoryagents such as suspending, stabilizing and/or dispersing agents.Alternatively, the active ingredient may be in powder form forconstitution with a suitable vehicle, sterile pyrogen-free water orother solvents, before use. Topical administration in the presentinvention may employ the use of a foam, gel, cream, ointment,transdermal patch, or paste.

[0100] Pharmaceutically acceptable compositions and methods for theiradministration that may be employ for use in this invention include, butare not limited to those described in U.S. Pat. Nos. 5,736,154;6,197,801 B1; 5,741,511; 5,886,039; 5,941,868; 6,258,374 B1; and5,686,102.

[0101] One preferred embodiment is the intracavemous administration ofabout 0.5 to 2.0 ml of the AAV-VEGF, AAV-BDNF, AAV-bFGF, AAV-PDGF,AAV-NT-3, AAV-NT-4, or AAV-Ang-1 gene therapy vector at a concentrationof about 10¹⁰ virus titer.

[0102] The magnitude of a therapeutic dose in the acute or chronictreatment of erectile dysfunction or female sexual arousal disorder willvary with the severity of the condition to be treated and the route ofadministration. The dose, and perhaps dose frequency, will also varyaccording to age, body weight, condition and response of the individualpatient. A preferred dosage for the treatment or prevention of maleerectile dysfunction and/or female sexual arousal disorder is about10-200 mcg/70 Kg body about once every two to six months.

[0103] One preferred embodiment of the present invention contemplatesthe administration by the application of a cream or by an injection tothe clitoris and vaginal wall of a patient with female sexual arousaldisorder in an amount sufficient to improve blood flow and regeneratenerve and smooth muscle in the clitoris and vaginal wall.

[0104] It should be noted that the attending physician would know how toand when to terminate, interrupt or adjust therapy to lower dosage dueto toxicity, or adverse effects. Conversely, the physician would alsoknow how to and when to adjust treatment to higher levels if theclinical response is not adequate (precluding toxic side effects).

[0105] Any suitable route of administration may be used. Dosage formsinclude tablets, troches, cachet, dispersions, suspensions, solutions,capsules, patches, and the like. See, Remington's PharmaceuticalSciences.

[0106] In practical use, VEGF, BDNF, bFGF, PDGF, NT-3, NT-4, or Ang-1peptides, proteins, polynucleotides, nucleic acids, or agent thatenhances production and/or erection or sexual arousal stimulatingfunction of said factor may be combined as the active in intimateadmixture with a pharmaceutical carrier or incipient according toconventional pharmaceutical compounding techniques. The carrier may takea wide form of preparation desired for administration, topical orparenteral. In preparing compositions for parenteral dosage form, suchas intravenous injection or infusion, similar pharmaceutical media maybe employed, water, glycols, oils, buffers, sugar, preservatives,liposomes, and the like known to those of skill in the art. Examples ofsuch parenteral compositions include, but are not limited to dextrose 5%w/v, normal saline or other solutions. The total dose of VEGF, BDNF,bFGF, PDGF, NT-3, NT-4, or Ang-1 to be administered may be administeredin a vial of intravenous fluid, ranging from about 1 ml to 2000 ml. Thevolume of dilution fluid will vary according to the total doseadministered.

[0107] The invention also provides for kits for carrying out thetherapeutic regimens of the invention. Such kits comprise in one or morecontainers therapeutically effective amounts of the VEGF, BDNF, bFGF,PDGF, NT-3, NT-4, Ang-1, or an agent stimulating the production and/orfunction of VEGF, BDNF, bFGF, PDGF, NT-3, NT-4, Ang-1, or somecombination thereof in pharmaceutically acceptable form. Preferredpharmaceutical forms would be in combination with sterile saline,dextrose solution, or buffered solution, or other pharmaceuticallyacceptable sterile fluid. Alternatively, the composition may belyophilized or dessicated; in this instance, the kit optionally furthercomprises in a container a pharmaceutically acceptable solution,preferably sterile, to reconstitute the complex to form a solution forinjection purposes. Exemplary pharmaceutically acceptable solutions aresaline and dextrose solution.

[0108] In another embodiment, a kit of the invention further comprises aneedle or syringe, preferably packaged in sterile form, for injectingthe composition, and/or a packaged alcohol pad. Instructions areoptionally included for administration of composition by a physician orby the patient.

[0109] In yet another embodiment, a kit of the invention furthercomprises in a container a pharmaceutically acceptable compositionsuitable for topical administration. Instructions are optionallyincluded for administration of composition by a physician or by thepatient.

[0110] The following examples are intended to illustrate but not tolimit the invention.

EXAMPLE 1 The Effect of Vascular Endothelial Growth Factor (VEGF) on aRat Model of Traumatic Arteriogenic Erectile Dysfunction

[0111] Animal Model

[0112] Fifty 3-months-old male Sprague Dowley rats weighing 350˜400grams were anesthetized with intraperitoneal pentobarbital (35 mg/kg)after being sedated with inhalation of methoxyflurane. Midlinelaparotomy was performed to identify the iliac vessels. Under operatingmicroscope, the iliac veins were carefully dissected to expose theinternal iliac arteries that vary from 2 to 4 in number. Rats in theexperimental group (n=44) underwent bilateral ligation of internal iliacarteries at their origin. A separate incision was made in the perineumto identify one of the crura. A 23-gauge scalp vein needle was insertedto the crus and connected to a pressure monitor for intracavernouspressure monitoring during electrostimulation of the cavernous nerve.Additional ligation of arterial branches was performed until minimal orno intracavernous pressure rise during electrostimulation.Intracavernous injection of 4 μg of VEGF in PBS (phosphate-bufferedsaline) with 0. 1% BSA (bovine serum albumin); 2 μg of VEGF in PBS with0. 1% BSA; or PBS solution with 0. 1% BSA was then administered throughthe same needle to 3 groups of rats (n=16, 12 and 16 respectively). Thewound was closed in layers and the animals were closely monitored for upto 6 weeks. At week 1, 2 and 6 roughly one-third of rats from each groupunderwent electrostimulation of the cavernous nerve to assess erectilefunction and then sacrificed. Penile tissues of 3 rats randomly chosenfrom each of the three subgroups at 2 and 6 weeks were obtained forimmunohistochemical staining and electron microscopic examination.

[0113] Electrical Stimulation

[0114] Bipolar platinum-wire electrodes were used to stimulate thecavernous nerve. The exposed end of the electrodes were hooked aroundthe nerve to be stimulated, with the positive electrode being positionedproximally and the negative electrode two to three mm distally. Stimulusparameters were 1.5 volts, frequency of 20 pulses per second, pulsewidth of 0.2 msec, and the duration of 50 seconds. Intracavernouspressure was monitored and recorded by inserting a number 23 scalp veinneedle to one of the crura and connected to a pressure monitor.

[0115] Immunohistochemical Staining

[0116] Penile tissue were fixed for 3 hours in a cold, freshly preparedsolution of 2% formaldehyde, 0.002% picric acid in 0.1 M. phosphatebuffer, pH 8.0. Tissues were cryoprotected for 24 hours in cold 30%sucrose in 0.1 M. phosphate buffer, pH 8.0. They were then embedded inO.C.T. compound (Tissue-Tek, Miles Laboratory), frozen in liquidnitrogen, and stored at −70° C. After freezing, Cryostat tissue sectionswere cut at 10 μm., adhered to charged slides, air-dried, and hydratedfor 5 min. with 0.05 M. sodium phosphate buffer (PBS, pH 7.4). Sectionswere treated with hydrogen peroxide/methanol to quench endogenousperoxidase activity. After rinsing with water, sections were washedtwice in PBS for 5 min. followed by 30 min. of room-temperatureincubation with 3% horse serum/PBS/0.3% triton X-100. After drainingsolution from sections, tissues were incubated for 60 min. at roomtemperature with mouse monoclonal anti-nNOS (Transduction Laboratories,Lexington, Ky.) at a 1:500 dilution. After washing for 5 min. withPBS/TX and then for 5 min. twice with PBS alone, sections wereimmunostained with the avidin-biotin-peroxidase method (Elite ABC,Vector Labs, Burlingame Calif.), with diaminobenzidine as the chromagen,followed by counterstaining with hematoxylin.

[0117] Electron Microscopy

[0118] The penis was dissected, thinly sliced (˜1 mm thick) and placedin Kamovsky's fixative (1% para-formaldehyde/3% glutaraldehyde/0.1 Msodium cacodylate buffer, pH 7.4) at room temperature for 30 minutes andthen stored at 4° C. The fixed tissue was then rinsed in buffer,post-fixed in 2% aqueous Os04, and stained en bloc with uranyl acetatebefore being dehydrated in ethanol, cleared in propyline oxide, andembedded in eponatel2 (Ted Pella Co., Redding, Calif.). Thick sectionswere cut and stained with toludine blue, examined under light microscopeto select the area to be thin-sectioned. Thin sections were cut by Leicaultracut E microtome (Bannockburn, Ill.), stained with uranyl acetateand Reynold's Lead to enhance contrast and examined under Philips Tecnai10 electron microscope (Eidhoven, Netherlands).

[0119] Statistics

[0120] Data were evaluated with Mann-Whitney rank-sum test. Significancewas defined as p<0.05.

[0121] Erectile Function

[0122] The peak sustained intracavernous pressure duringelectrostimulation of the cavernous nerve is shown in Table I. There isno difference in intracavernous pressure between the sham operated andthe normal rats. After bilateral ligation of the internal iliacarteries, the intracavernous pressure immediately dropped to around 20cm H₂O and produced no or minimal pressure increase in response toneurostimulation in all rats. In the PBS-treated group, poor erectileresponse persisted at weeks 1 and 2 and slight recovery of erectilefunction was noted at week 6.

[0123] In the VEGF-treated rats, at weeks 1 and 2, moderate recovery oferectile function was noted in the 4-μg group but not the 2-μg group. Atweek 6, statistically significant improvement in intracavernous pressurewas seen in both the 2-μg and 4-μg groups as compared with thePBS-treated group. The intracavernous pressure of the 4-μg group wasalso significantly higher than that of the 2-μg group.

[0124] To identify the new source of blood flow in the 4-μg VEGF-treatedgroup, we noted a decrease in erectile response after clamping oneexternal iliac artery and no erectile response at all after clampingboth external iliac arteries. This strongly suggests that the collateralvessels were derived from the external iliac arteries. TABLE 1 Peaksustained intracavernous pressure (cm H₂O) during electro- stimulationof the cavernous nerves in saline- and VEGF-treated rats Saline treatedGroup VEGF-treated Group PBS + 0.1% BSA PBS + 2 μg VEGF PBS + 4 μg VEGFWeek 1 20.33 ± 3.45 23.50 ± 2.38 71.17 ± 16.89 (n = 6) (n = 4) (n = 6)Week 2 27.75 ± 9.70 43.00 ± 8.37 86.25 ± 8.18 (n = 4) (n = 4) (n = 4)Week 6 46.75 ± 14.85 69.00 ± 8.83 96.67 ± 13.50 (n = 6) (n = 4) (n = 6)

[0125] Histochemistry

[0126] There was a trend of decreased nNOS-immunoreactive in both dorsaland intracavemous nerves two weeks after arterial ligation in allsubgroups. At week 6, moderate recovery of nNOS-positive nerve fiberswas noted in both dorsal and intracavemous nerves in both 2 & 4 μgVEGF-treated groups but not in the PBS-treated group. However, thedifferences were not statistically significant by computer-assistedimage analysis.

[0127] Electron Microscopy

[0128] Dorsal Nerve:

[0129] In sham-operated rats, no difference was noted between specimensobtained from the 2 and 6-week groups. The dorsal nerve in these ratswas filled with both myelinated and non-myelinated nerve bundles. Themean diameter of the individual myelinated nerve axon was 4.42±1.36 μm(excluding myclin sheath). The mean thickness of the myelin sheath was0.58±0.21 μm. The mean diameter of the non-myelinated nerve fibers was0.96±0.37 μm. The nuclei of Schwann cells were seen occasionally nearthe nerve fibers.

[0130] In ligated +PBS treated rats, dramatic changes were noted at week2. There was an increase in the number of Schwann cells and many ofwhich contained vacuous within the cytoplasm. There was also a decreasein the number of both non-myelinated and myelinated nerve fibers.Overall the size of the axons was smaller than that of the controls, andmany of the non-myelinated nerve fibers were smaller and less discrete.At week 6, varying degree of regeneration of both myelinated andnon-myelinated nerve fibers was apparent. However, the nerve fibers ofboth the myelinated and non-myelinated nerve fibers were still smallerthan the control groups. (mean myelinated axon 3.17±1.01 μm, myelinsheath 0.46±0.11 μm, non-myelinated axon 0.81±0.38 μm, p=0.062, 0.189,and 0.069 respectively compared with those of the sham group.) Many ofthe non-myelinated fibers were less than one third of the size of thelarger ones. There was also an increase in the number of nucleatedSchwann cells.

[0131] At week 6, in ligated rats treated with 4 μg of VEGF, the meandiameter of the myclinated axons (6.19±2.38 μm) was larger than theligated +PBS treated ones (3.17±1.01 μm) with a p=0.008. The meandiameter of the nonmyelinated axons was 0.82±0.45 μm which was similarto that of the ligated +PBS treated rats (0.81±0.38 μm). However, thenon-myelinated nerve fibers appeared more even in size.

[0132] Intracavernosal Smooth Muscle:

[0133] In sham-operated rats, no difference was noted between thespecimens from the 2 and 6-week groups. The smooth muscle cells, most ofwhich were arranged in clusters, were embedded in fine strands offibro-connective tissue. The cytoplasm of these myocytes containedabundant contractile myofilaments and dense bodies. Occasionally, smallaggregates of organelles, including mitochondria, rough endoplasmicreticulum and Golgi apparatus, were found adjacent to the nucleus. Thecell membrane (sarcolemma) was consisted of many alternating dense andlight bands with the latter containing numerous pinocytotic vesicles(caveolae). The intercellular spaces were narrow and many cell-cellcontacts (gap junctions) were visible. Nerve terminal varicosities wereseen occasionally near clusters of smooth muscle cells.

[0134] At week 2, in ligated +PBS-treated rats, many atrophic smoothmuscle cells separated by large amounts of collagen fibers were noted.These atrophic smooth muscle cells appeared scattered in a sheet ofconnective tissues while normal smooth muscle cells were separated onlyby minimal amounts of connective tissues. At week 6, the number ofnormal-appearing smooth muscle cells increased, although many of themstill showed significant loss of myofilaments.

[0135] In ligated +4 μg of VEGF-treated rats (both 2 and 6-week groups),most of the smooth muscle cells appeared normal with large amount ofmyofilaments and narrow intercellular spaces.

[0136] Endothelium:

[0137] In sham-operated rats, the cavernous sinusoids were lined withintact endothelium, the cytoplasm of which contained numerouspinocytotic vesicles (caveolae), mitochondria, rough endoplasmicreticulum, and Golgi apparatus. The nuclei of the endothelial cells weresparsely seen and appeared flattened. In ligated +PBS-treated rats,(both 2 and 6-week groups) the capillary and cavernous sinusoidalendothelium appeared normal, although increase in the number ofendothelial cells could be seen in some fields. In ligated +4 μg ofVEGF-treated rats, (both 2 and 6-week groups) striking differences werenoted when compared with other groups of rats. Many reactive endothelialcells with plump nuclei could be seen lining the sinusoids andcapillaries. These endothelial cells were larger and more numerousindicative of endothelial cell hypertrophy and hyperplasia. Lee M C, etal., J. Urol. 167:761-7 (2002).

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[0145] 8. Leung, D. W., et al., Vascular endothelial growth factor is asecreted angiogenic mitogen. Science, 1989. 246(4935): p. 1306-9.

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[0147] 10. Hopkins, S. P., et al., Controlled delivery of vascularendothelial growth factor promotes neovascularization and maintains limbfunction in a rabbit model of ischemia. Journal of Vascular Surgery,1998. 27(5): p. 886-94; discussion 895.

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[0149] 12. Hariawala, M. D., et al., VEGF improves myocardial bloodflowbut produces EDRF-mediated hypotension in porcine hearts. Journal ofSurgical Research, 1996. 63(1): p. 77-82.

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[0151] 14. Bauters, C., et al., Physiological assessment of augmentedvascularity induced by VEGF in ischemic rabbit hindlimb. AmericanJournal of Physiology, 1994. 267(4 Pt 2): p. H1263-71.

[0152] 15. Takeshita, S., et al., Therapeutic angiogenesis. A singleintraarterial bolus of vascular endothelial growth factor augmentsrevascularization in a rabbit ischemic hind limb model. Journal ofClinical Investigation, 1994. 93(2): p. 662-70.

[0153] 16. Takeshita, S., et al., Intramuscular administration ofvascular endothelial growth factor induces dose-dependent collateralartery augmentation in a rabbit model of chronic limb ischemia.Circulation, 1994. 90(5 Pt 2): p. 11228-34.

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[0156] 19. Baumgartner, I., et al., Constitutive expression of phVEGF165 after intramuscular gene transfer promotes collateral vesseldevelopment in patients with critical limb ischemia [see comments].Circulation, 1998. 97(12): p. 1114-23.

[0157] 20. Losordo, D. W., P. R. Vale, and J. M. Isner, Gene therapy formyocardial angiogenesis. American Heart Journal, 1999. 138(2 Pt 2): p.132-41.

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[0159] 22. Kim, J. H., et al., Experimental hypercholesterolemia inrabbits induces cavernosal atherosclerosis with endothelial and smoothmuscle cell dysfunction. Journal of Urology, 1994. 151(1): p. 198-205.

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EXAMPLE 2 Intracavernosal Vascular Endothelial Growth Factor (VEGF) andAdeno-Associated Virus Mediated VEGF Gene Therapy Prevents and ReversesVenogenic Erectile Dysfunction in Rats

[0170] Animal Groups

[0171] Male Sprague-Dawley rats age 3-6 months (wt 350-450 grams) wereused in this study. They were housed in our animal care facility withrat chow and water available ad libitum on a 12 hr light/dark cycle. Allanimal care, treatments and procedures were performed in compliance withrequirements of the Committee on Animal Research at our institution.Rats were randomly divided for the animal model of vasculogenic ED(Experiment 1) and the VEGF prevention trial (Experiment 2). For theVEGF treatment trial (Experiment 3), the animals underwent castrationand then were treated with VEGF after venous leak was demonstrated, toevaluate the efficacy of VEGF treatment in reversing establishedvenogenic ED.

[0172] Experiment 2:

[0173] To determine normal values for rodent pharmacologiccavernosometry and validate the model of venogenic ED in the rat,vasculogenic ED was induced. Arterial insufficiency was produced afterperforming a bilateral ligation of the internal iliac arteries. Theacute and chronic effects of arterial insufficiency were evaluated 7days and 30 days after bilateral iliac artery ligation (n=7). VenogenicED was induced by castration, and pharmacologic cavernosometry wasperformed 6 weeks after surgery (n=10). Control animals underwent a shamlaparotomy and studied 6 weeks later (n=13).

[0174] Experiment 2:

[0175] For the prevention trial of VEGF in rats with venogenic erectileED, adult males were castrated and immediately treated with hormone,intracavernosal VEGF. Hormone replacement was accomplished using asubcutaneously placed testosterone-filled silastic implant (n=6), aspreviously described¹⁷. A therapeutic testosterone serum titer wasconfirmed in this animal group by testosterone radioimunoassay performedby the biomedical core lab at our institution¹⁸. Intracavemous treatmentwith VEGF was administered using either recombinant VEGF protein (n=5)or an adeno-associated virus vector expressing the VEGF gene (AAV-VEGF,n=14). Control animals received an silastic implant containing saline(n=3), an intracavernous injection of normal saline (n=3) or anadenovirus transfection vector expressing lacZ reporter gene without theVEGF gene (AAV-LacZ, n=11).

[0176] Experiment 3:

[0177] The trial of VEGF treatment was performed in castrated animalsthat were shown, 6-8 weeks following castration, to develop venogenicerectile ED by pharmacologic cavernosometry. These animals (n=8) weretreated with intracavernous VEGF protein and then after one monthcavernosometry repeated to measure the effect of VEGF treatment.

[0178] Animal Treatments

[0179] Surgical Preparation:

[0180] Prior to all surgical procedures, animals received anesthesiaconsisting of isoflurane inhalation as pre-anesthetic followed by anintraperitoneal injection of sodium pentobarbital (40 mg/kg). After theanimal was asleep, electric clippers were used to trim the ventralabdominal hair and the skin was prepped with clorhexidine scrub.Antiseptic technique was maintained for all procedures. Followingsurgery, the anterior abdominal fascia and skin were approximated with4-0 silk suture, analgesic buprenorphine (0.5 mg/kg SC) was administeredand the animal allowed to awaken covered with a heating pad. Euthanasiawas accomplished by an intraperitoneal injection of sodium pentobarbital(200 mg/kg) followed by bilateral thoracotomy when the animal was fullyasleep.

[0181] Arterial Ligation Surgery:

[0182] A 2 cm midline longitudinal low abdominal incision was made and awheatlander retractor placed so that the plane between the prostate andsigmoid colon could be bluntly opened. A dissecting microscope with2.5-10× objectives was essential to safely performing this procedure.Using sterile cotton-tipped swabs, the iliac artery bifurcation wasidentified and the common iliac exposed to the external iliac take-off.The internal iliac arteries were identified as those medial branches offthe common iliac between the iliac bifurcation and the take-off of theexternal iliac. These were doubly ligated using 7-0 nylon sutures. Afterthis was performed on both the right and left side, the incision wasclosed and the animal recovered as noted above.

[0183] Castration:

[0184] A 2 cm midline longitudinal low abdominal incision was made andeach testicle was grasped using forceps and brought into the incision.Each gubernaculum was divided using electrocautery and then thespermatic cords ligated with 4-0 silk suture and divided. Afterconfirming hemostasis, the abdomen was closed and the rat recovered asabove.

[0185] Intracorporal Injections:

[0186] A 1.5 cm oblique incision was made in the lower abdominal skinextending from the midline just above the penile hilum to below thelevel of the glans about 1 cm lateral to the midline. The skin wassharply dissected from the anterior surface of the penis and then thepenis was retracted anteriorly using a towel clamp placed around itatraumatically with the foreskin left intact. Using blunt dissection thepenile base and crura were exposed. The ischiocavernosus muscles weresharply dissected off the anterior surface of the crus until the whiteof the tunica albuginea of the corpora cavernosa was identified. Thecrus was then gently cannulated using a 23 gage butterfly needle, andsaline flush with a visual erectile response was used to confirm thatthe needle tip was truly intracavernosal. The intracavernosal injectionswere then administered with either VEGF protein (Calbiochem, La Jolla,Calif. #676472) at the dosage 4 ug/injection (in 0.1 cc PBS with 0.1%BSA), AAV-VEGF (10¹⁰ viral particles in 0.1 cc NS), AAV-LacZ (10¹⁰ viralparticles in 0.1 cc NS), or 0.1 ml NS alone. The AAV-VEGF and AAV-LacZconstructs were a generous gift from Dr. Yuet W. Kan (Howard HughesMedical Research Institute, San Francisco, Calif.)^(19,20). Followinginjection, the needle was left in place for 5 minutes and then removedto allow the medication to diffuse throughout the cavernosal space.Immediately thereafter, pinpoint electrocautery was applied to theneedle hole for hemostasis and then the wound was closed and the animalrecovered as above.

[0187] Testosterone Replacement:

[0188] Following castration, testosterone- or saline-filled silasticimplants were placed in the subcutaneous tissue of the anteriorabdominal wall, as previously described¹⁷. Implants were prepared usingsterile silastic tubing (Dow Corning, Midland, Mich. #602-265, innerdiameter 0.062″) that was filled with testosterone propionate powder(Sigma Chemical, St. Louis, Mo.) with the aid of wall suction. Byradioimmunoassay¹⁸, serum testosterone titer was found to beundetectable in the castrated animals and in the normal range foranimals given testosterone implants.

[0189] Pharmacologic Cavernosometry in the Rat:

[0190] To perform pharmacologic cavernosometry, both the right and leftcrura were separately cannulated using 23 gauge butterfly needles asdescribed above. One cannula was flushed with sterile heparinized saline(100U heparin/ml NS) and attached to a pressure detector for continuousintracorporal pressure (ICP) monitoring as previously described¹¹. Thecontralateral cannula was attached to an infusion pump (Harvard Pump,Southwick, Mass. #55-2222), filled with sterile dilute heparinizedsaline (20U heparin/ml NS). The baseline ICP was recorded (flaccid ICP)and then a dose of papavarine (1 mg in 0.1 cc NS) was administeredthrough the infusion cannula. Five minutes was allowed for thepapavarine to diffuse throughout the corpora and then the infusioncannula was flushed with heparinized saline and the pressure monitorcannula vented to normalize ICP after flushing. After 5 minutes more,the ICP was again recorded (ICP after papavarine) and the infusionstarted. An infusion rate of 0.05 ml/min was started and increased (by0.05 ml/min every 10 seconds) until the ICP started to rise. Subsequentincreases in inflow rate were made only after the ICP reached a plateaupressure. By slowly adjusting the inflow rate, an intracorporal pressureof 100 cm H₂O (erectile pressure) was reached and the infusion raterequired to maintain this pressure recorded (the maintenance rate).After this pressure was steady for 20 seconds, the infusion wasterminated and the change in ICP over the subsequent 60 seconds wasrecorded (the drop rate).

[0191] Tissue Preparation:

[0192] After pharmacologic cavernosometry was performed, the penis wasamputated at the crural bony attachments and immediately placed inice-cold saline. The Y-shaped crura was sharply cut from the penile baseand then a 1 mm thick slice cut for electron microscopy and placed inKamofsky's solution (3% gluteraldehyde, 1% para-formaldehyde, 0.1 Msodium cacodylate buffer, pH 7.4). A 3 mm thick section of the distalpenile shaft was then cut and placed in 10% normal buffered formalin forparaffin sections, and the balance of the penile shaft was flash frozenusing dry ice in OCT compound (Sakura Finetek USA, Torrance, Calif.) forfrozen sectioning and immunohistochemistry.

[0193] Immunohistochemistry:

[0194] Frozen sections were cut at 10 microns, adhered to chargedslides, air dried for 15 minutes then rehydrated with 0.05M PBS for 5minutes. Sections were treated with hydrogen peroxide/methanol to quenchendogenous peroxidase activity. After rinsing, sections were washedtwice in PBS for 5 minutes then incubated with 3% horse serum and 0.3%triton X-100 at room temperature for 30 minutes. The serum solution wasdrained and then sections were incubated for 60 minutes with mousemonoclonal anti-alpha-smooth muscle actin (Sigma, St. Louis, Mo.) at adilution of 1:4000 in PBS. After washing, sections were immunostainedusing the avidin-biotin-peroxidase method (Elite ABC-Vector Labs,Burlingame, Calif.), with diaminobenzidine as the chromogen, followed bycounterstaining with hematoxylin. Immunochemistry was performed inpenile tissues from 4 rats randomly chosen from each subgroup.

[0195] Enzyme-Linked Immuno-Sorbent Assay:

[0196] Serum samples from both systemic and penile blood were collectedafter whole-blood centrifugation. Solid phase enzyme-linkedimmuno-sorbent assay for VEGF was performed using the Quantikine M mouseVEGF Immunoassay Kit (R&D Systems, Minneapolis, Minn.) as previouslydescribed²¹. Briefly, samples were diluted and added to micro platestrip wells that were then treated with the enzyme-labeledimmunoreactant VEGF conjugate. After incubation for 2 hours and washing,the substrate solution was added and incubated for 30 minutes. The stopsolution was added and then the optical density in each well determinedusing a micro plate reader set to 450 nm. Sample results were plotted ona curve generated by the optical density of standard samples ranging inconcentration (0-500 pg/ml VEGF).

[0197] Transmission Electron Microscopy:

[0198] After fixing in Karnofsky's solution for 30 minutes at roomtemperature, 1 mm sections at the penile base were stored at 40 C untilprocessed, as previously described 22. Briefly, samples were rinsed inPBS, post-fixed in 2% aqueous OsO4 and stained en bloc with uranylacetate. They were then dehydrated in ethanol, cleared in propylineoxide and embedded in Eponate 12 (Ted Pella Co., Redding, Calif.). Thicksections were cut and stained with toluidine blue to select specificareas for thin sectioning. Thin sections were cut, stained with bothuranyl acetate and Reynold's Lead, and examined under the PhillipsTecnai 10 transmission electron microscope. Penile tissues from 4randomly chosen rats in each subgroups were subjected to electronmicroscopic examinations.

[0199] Statistical Analysis:

[0200] Data in the present studies was analyzed using the student'st-test (homoscedastic, 2-tailed) when 2 means were compared (Experiment1 and 2). A paired, 2-tailed t-test was used where values representfindings before and after treatment in the same animals (Experiment 3).

Results

[0201] Experiment 1

[0202] Model Validation:

[0203] The first goal was to determine normal values for pharmacologiccavernosometry in a rat model of vasculogenic ED. As shown in Table 2,flaccid ICP were comparable, in the range of 30 cm H2O. After papavarineinjection, however control animals had a steep rise in ICP to >100 cmH₂O while the castrated and ligated animals had less response. Only aminimal increase in ICP (5-10 cm H₂O) was noted in the animals followingeither castration or chronic internal iliac ligation, characteristic ofvasculogenic ED. Acute ligation animals had a better response topapavarine yet significantly less than seen in normal animals. After theinfusion was started, both the control and acute ligation group promptlyachieved erectile pressure with minimal inflow required. When theinfusion was stopped, these animals had a minimal pressure drop,evidencing their intact veno-occlusive mechanism. The castration andchronic ligation groups, on the other hand, required a significantlyhigher infusion rate to maintain erectile pressure and experienced asteep pressure drop when the infusion was terminated. These findings arecharacteristic of venous leakage in the chronic ligation and castrationgroups. TABLE 2 (Experiment 1) Cavernosometric findings in rat model ofvasculogenic ED. ICP After Maintenance Drop Rate in Flaccid ICPPapavarine Rate 1 min. (cm H₂O) (cm H₂O)* (ml/min)* (cm H₂O)* Control35.4(+/−9.3) 104(+/−59) 0.024(+/−0.3) 9(+/−13) Castration 22.0(+/−5.2)35.0(+/−5.0) 1.14(+/−0.5) 75(+/−5.4) Acute 28.7(+/−7.6) 73.3(+/−10)0.06(+/−0.12) 13.2(+/−13.8) Ligation Chronic 29.3(+/−7.6) 38.3(+/−19)1.9(+/−1.8) 45.8(+/−19) Ligation

[0204] Experiment 2

[0205] Prevention Trial:

[0206] Our second goal was to perform a prevention trial usingintracavernosal VEGF either in the form of recombinant protein orvirus-directed gene expression vector in an attempt to prevent thedevelopment of venogenic erectile ED in castrated animals. As shown inTable 3, flaccid ICP was again in the range of 30 cm H₂O in each of theanimal groups. After papavarine administration, however, both controlgroups (castration only and castration with LacZ injection) exhibitedonly a weak rise in ICP (5-10 cm H₂O), required a significant infusionrate to sustain an erectile ICP of 100 cm H₂O, and had a steep pressuredrop after the inflow was terminated. In contrast, the 3 treatmentgroups exhibited nearly normal erectile function with high ICP inresponse to papavarine, a very low maintenance rate to sustain erectileICP and minimal pressure drop when the infusion was stopped. Of note,the VEGF gene-treated animals showed marginally less erectile functionwith a lesser response to papavarine and a higher drop rate than theanimals treated with either testosterone replacement or intracavernosalVEGF protein. TABLE 3 (Experiment 2) Cavernosometric findings incastrated animals after prevention trial of testosterone replacement(C + Testosterone), VEGF protein treatment (C + VEGF), AAV − VEGF genetherapy (C + VEGF gene), or LacZ control (C + LacZ control). ICP AfterMaintenance Drop Rate in Flaccid ICP Papavarine Rate 1 min. (cm H₂O) (cmH₂O)* (ml/min)* (cm H₂O)* C + saline 23.4(+/−5.3) 29.4(+/−15)0.51(+/−0.26) 56.1(+/−15) C + 28.9(+/−7.5) 87.7(+/−26) 0.09(+/−0.1)11.7(+/−16) Testo- sterone C + 27.8(+/−5.2) 85.0(+/−28) 0.04(+/−0.09)9.0(+/−20) VEGF C + 23.4(+/−7.1) 61.4(+/−36) 0.04(+/−0.03) 27.8(+/−18)VEGF gene C + LacZ 27.0(+/−6.7) 36.0(+/− 0.25(+/−0.31) 58.6(+/−8.1)12.8)

[0207] Experiment 3

[0208] Treatment Trial:

[0209] The third phase was to perform a treatment trial usingintracavernosal VEGF in animals with venous leak. The goal was to assessthe efficacy of VEGF at reversing established venogenic erectiledysfunction in an animal model. Animals were castrated and then 4-6weeks later underwent cavernosometry. As shown in Table 4, before VEGFtreatment, this animal group displayed a weak response to papavarinewith intracorporal pressure reaching 33 cm H₂O compared to normalanimals who attain nearly 100 cm H₂O with such treatment (see Table 2).Also, these castrates required a relatively high maintenance rate (0.19ml/min.) to achieve erectile pressure and a steep drop rate when theinfusion was terminated (45.1 cm H₂O in 60 seconds), evidencing venousleak. After these animals received intracorporal VEGF treatment,however, nearly normal erectile function returned with a prompt rise inintracorporal pressure after papavarine (to 84 cm H₂O), a lowmaintenance rate (0.08 ml/min.) to achieve erectile pressure and aminimal drop in intracorporal pressure (17.4 cm H₂O in 60 seconds) afterthe infusion was terminated. TABLE 4 (Experiment 3) Cavernosometricfindings in VEGF treatment trial. ICP After Maintenance Drop Rate inFlaccid ICP Papavarine Rate 1 min. (cm H₂O) (cm H₂O)* (ml/min)* (cmH₂O)* Before VEGF 22.4(+/−6.9) 33.0(+/ 0.19(+/ 45.1(+/−18) treatment (6−12.3) −0.18) wks after castration) 1 month 25.3(+/−8.5) 83.9(+/−31)0.08(+/− 17.4(+/−24) following 0.15) VEGF treatment

[0210] Animals were castrated and then shown to have venous leak (afterapproximately 6 weeks) by pharmacologic cavernosometry. They were thentreated with intracavernosal VEGF and one month later underwent repeatcavernosometry. (* p<0.05)

[0211] Immunohistochemistry:

[0212] FIGS. 1A-D represent cross-sectional micrographs of the rat penisat the proximal shaft, after immunohistochemistry for alpha actin. Alphaactin, a marker for penile smooth muscle, stains brown and can be seensurrounding the sinusoidal spaces. Qualitatively, we see decreasedsmooth muscle content in FIG. 1B (6 weeks after castration) compared toFIG. 1A (normal control). In castrates treated with either testosteronereplacement (FIG. 1C) or intracavernosal VEGF protein (FIG. 1D) thequantity of smooth muscle returns to normal morphology. Computerizedimage analysis with Adobe Photoshop was used to quantify the area ofimmunostaining by counting the number of digitized pixels correspondingto the area of brown staining, thereby providing some numericalcomparison of the quantity of smooth muscle in each specimen. Thisanalysis shows the following pixel count: sham (43518), castrated(37214), AAV-VEGF treated (51690), VEGF protein treated (52990).

[0213] Transmission Electron Microscopy

[0214] Dorsal Nerve:

[0215] In sham-operated rats (FIG. 2A), the dorsal nerve was filled withboth myelinated and non-myelinated nerve bundles. The mean diameter ofthe individual myelinated axon (excluding myelin sheath) was 2.54±1.04μm. The mean thickness of the myelin sheath was 0.74±0.21 μm. The meandiameter of the non-myelinated axon was 0.97±0.35 μm. The cytoplasm andnuclei of Schwann cells were seen occasionally near the nerve fibers.

[0216] In castrated rats, with or without LacZ injection (FIG. 2B), thediameter of both the myelinated and non-myelinated axons appearedsmaller than those of the sham-operated rats. Mean diameters were thefollowing: myelinated axon 1.64±1.0 μm; myelin sheath 0.49±0.13 μm;non-myelinated axon 0.64±0.32 μm. Comparing the castrated rats to thesham-operated rats, the p values were 0.06, 0.004 and 0.001respectively. Many non-myelinated nerve fibers became indistinct andsmaller. There was also an increase in the number of nucleated Schwanncells.

[0217] Although many small myelinated nerve fibers were still present incastrated rats treated with VEGF or AAV-VEGF (FIG. 2C), larger fiberswith thick myelin sheaths were also noted. The mean diameter of themyelinated nerve and myelin sheath were 2.36±0.92 μm and 0.93±0.44 μmrespectively. The non-myelinated nerve fibers were more clearly definedbut were not as abundant as the sham group. The mean diameter ofnon-myelinated axons was 0.96±0.33 μm. Comparing the VEGF-treated groupto the castrated +Lac Z group, the p values of myelinated axon, myelinsheath and nonmyelinated axon were 0.113, 0.05 and 0.000 respectively.The nerve fibers and myelin sheath in the testosterone replacement groupappeared similar to the sham group.

[0218] Intracavernosal Tissues

[0219] Intracavernous Smooth Muscle Cells:

[0220] In sham-operated rats, the smooth muscle cells (myocytes) wereusually arranged in clusters and were separated by fine strands offibroconnective tissue (FIG. 3A). The cytoplasm of these myocytescontained abundant contractile myofilaments and dense bodies.Occasionally, small aggregates of organelles, including mitochondria,rough endoplasmic reticulum and Golgi apparatus, were found adjacent tothe nucleus. The cell membrane (sarcolemma) consisted typically ofalternating dense bands and light bands. The light bands containnumerous pinocytotic vesicles (caveolae). The intercellular spaces amongmyocytes were usually quite narrow with many gap junctions connectingindividual cells. Nerve terminal varicosities were frequently seenlocated near clusters of smooth muscle cells. In low power micrographs(6,500×) of castration with or without LacZ rats, the smooth musclecells appeared scattered in a field of connective tissues (FIG. 3B). Themajor differences between the castrated and castrated +testosterone-treated rats were the increase in cytoplasmic myofilamentsand the decrease in intercellular spaces in the latter group of rats.The myocytes in testosterone-treated rats appeared packed in clustersrather than scattered.

[0221] Striking differences were noted when comparing the AAV-VEGF andVEGF protein-treated rats to the castrated +Lac Z rats. The smoothmuscles were arranged in clusters with minimal intercellular spaces(FIG. 3C). Under high power (9,400×), we noted the following: anincrease in myofilaments and dense bodies, a decrease in dense bands,and an increase in the number of caveolae within the light bands of thesarcolemma (FIGS. 3D &E).

[0222] Endothelial Cells:

[0223] In sham-operated rats (FIG. 4A), the cavernous sinusoids werelined by intact endothelium, the cytoplasm of which contained numerouspinocytotic vesicles (caveolae), mitochondria, rough endoplasmicreticulum, and Golgi apparatus. The nuclei of the endothelial cells wereoccasionally seen and appeared oval-shaped or elongated. In castrationwith or without LacZ rats, the appearance of the capillaries andcavernous sinusoidal endothelium was similar to the sham operated group.In AAV-VEGF and VEGF protein-treated rats, the nuclei of the endothelialcells lining most of the capillaries and sinusoids were plump and morenumerous, indicative of endothelial hypertrophy and hyperplasia (FIGS.4B&C).

[0224] Enzyme-Linked Immuno-Sorbent Assay:

[0225] The goal of using an adenovirus vector for delivering the VEGFgene is to transfect the penile tissue such that VEGF protein expressionmay be increase in the penis. To document that the AAV-VEGF treatedanimals had increased VEGF expression in the penile blood, samples weretaken from the penis (penile bleed following glans amputation) andcompared with a sample of systemic blood (from the abdominal aorta) foranimals groups treated with both AAV-VEGF (n=7) and AAV-LacZ (n=7)(Table 5). While the mean VEGF titer in the systemic serum of animalsthat did not receive the VEGF gene (AAV-LacZ group) is 9.5±12.6 pg/ml,the AAV-VEGF treated animals demonstrated a marked increase in VEGFtiter at 23.3±9.6 pg/ml (p=0.04). Similarly, serum from the penile bloodin the AAV-LacZ group had a VEGF titer of 13.6±11.4 pg/ml compared to amean of 29.7±14.4 pg/ml in the group receiving the intracavernosal VEGFgene (p=0.039). This difference is statistically significant suggestingthat increased VEGF expression is occurring in the penile tissue aftertreatment with AAV-VEGF. TABLE 5 Results of ELISA for VEGF protein (meanVEGF in pg/ml) in serum samples from animals treated with intracorporalAAV-VEGF (p = 0.03) and AAV-LacZ (p = 0.07) AAV-VEGF-treatedAAV-LacZ-treated animals animals Penile bleed 29.7 (+/−14.4) 13.6(+/−11.4) Systemic blood 23.3 (+/−9.6)  9.5 (+/−12.6)

Discussion

[0226] To test our hypothesis, we first developed an animal model ofvenogenic erectile ED (Experiment 1: Model validation). Mills etal^(10, 11, 26) have previously shown that rats develop venogenicerectile ED within 6-8 days following castration. Animals receivingtestosterone repletion maintain an intact veno-occlusive mechanism aftercastration. These studies were performed using ganglionicelectro-stimulation to generate an erection and the penile responsegauged with ICP monitoring during either cavernosometry¹¹ or a penilearterial inflow measurement using a laser Doppler flow¹⁰. Our goal wasto devise a technique for evaluating venous leak in animals similar tothe technique used in humans. For this reason, erection was generatedusing pharmacologic agents (papavarine) instead of ganglionicelectro-stimulation. The physiologic parameters (maintenance inflow rateand ICP drop rate) used to diagnose venous leak in humans^(27,28)werereproduced in a rat model. Using this technique, pharmacocavernosometricfindings were determined in normal animals and animals with venogenicand arteriogenic ED. This method was found to be a sensitive andreproducible technique to evaluate penile arterial insufficiency andvenous leak in a rat model.

[0227] This model was then used to evaluate the efficacy of VEGF,administered intracorporally as recombinant protein or adeno-associatedvirus gene vector, to prevent the development of venogenic erectile ED(Experiment 2: Prevention trial). It has been previously shown thatcastration induces an involution of the prostate gland and it'svasculature¹³. Furthermore, after testosterone replacement, endothelialcell proliferation is stimulated and both blood flow and vascularvolumes are normalized. After castration, prostatic VEGF synthesis isdown regulated, as determined by RT-PCR, western blot andimmunohistochemical analysis¹² Also, testosterone induces VEGFsynthesis, suggesting that VEGF may be a tissue mediator of androgeniceffects on the prostate. The goal of Experiment 2 was to determine ifVEGF could prevent the development of venous leak in the rat model. Bothtestosterone replacement and VEGF treatment maintained erectile functionwhen administered immediately after castration. Animal groups receivingno testosterone replacement or intracorporal AAV-LacZ showed persistentvenogenic erectile ED after castration. Histological examination ofsmooth muscle content and morphology revealed deterioration in both thequality and quantity of penile smooth muscle after castration. Electronmicroscopic examination also revealed alteration of cell membrane andwidening of intercellular spaces. Smooth muscle content as measured byalpha actin staining was normalized in animals receiving eithertestosterone or VEGF, evidence of preserved smooth muscle integrity withsuch preventative treatment.

[0228] The final phase was a treatment trial (Experiment 3) in whichanimals were first documented to have venous leak, 6 weeks aftercastration, and then treated with intracorporal VEGF protein. One monthafter such treatment, cavernosometry was repeated and restoration ofnear normal erectile function was found. We believe that this is thefirst experimental evidence of any medical therapy improving venogenicerectile ED. The exact mechanism by which VEGF improves erectilefunction is unknown. Nevertheless, we observed clear evidence ofrestoration of neural and smooth muscle integrity as well as hyperplasiaand hypertrophy of endothelial cells after VEGF treatment. Conceivably,increased cavernosal neovascularity may lead to functional or structuralchanges in the nerve and smooth muscles. Alternatively, the directeffect of VEGF on the nerve and smooth muscle may also play a role sinceVEGF has been reported to have a direct trophic effect on the penilesmooth muscle cells and spinal neurons in culture 14-16.

[0229] Nerve function and NOS expression is depressed with androgenablation, and this may be another target for VEGF action in the penis.Further studies are underway to study the mechanism of VEGF action inthe penis.

Conclusion

[0230] The technique presented here for pharmacologic cavernosometry isa simple and reproducible method to evaluate vasculogenic ED in a ratmodel. Normal erectile function and ED due to arterial insufficiency orvenous leak may be diagnosed by characteristic cavernosometric findings.Using this technique, the presence of veno-occlusive disease may bediagnosed in animals 6 weeks after either castration or ligation of theinternal iliac arteries.

[0231] Animals treated with testosterone replacement at the time ofcastration retain normal erectile function while those withouttestosterone replacement develop venous leak. If these animals aretreated with intracavernosal recombinant VEGF protein or AAV-VEGF at thetime of castration, their erectile function is maintained and venousleak is prevented. The mechanism for this is not known at present, butwe find a decrease in penile smooth muscle content in the castratedgroup compared with either the testosterone replacement group or thegroup receiving intracavernosal VEGF or AAV-VEGF. Penile smooth musclemorphology is uniformly degenerated after castration. Animals treatedwith the intracorporal AAV-VEGF transfection vector demonstratesignificantly more VEGF protein in their penile serum compared to thesystemic serum, and markedly more that control animals, indicating anincreased expression of penile VEGF in these animals.

[0232] When rats with established venogenic ED are treated with one doseof intracavernosal recombinant VEGF protein, their erectile functionreturns to nearly normal, with reversal of the veno-occlusive defect.Electronmicroscopy revealed endothelial cell hyperplasia and hypertrophyas well as restoration of smooth muscle and neural integrity in thepenile tissue after VEGF treatment. Since impairment of erectile nerve,endothelial cell, and the cavernous smooth musculature is the finalcommon pathway of various type of organic ED, VEGF therapy may hold thekey to prevention and cure of many forms of ED.

REFERENCES

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[0238] 6. Meisel, R. L., O'Hanlon, J. K. & Sachs, B. D.: Differentialmaintenance of penile responses and copulatory behavior by gonadalhormones in castrated male rats. Horm Behav, 18:, 56, 1984.

[0239] 7. Zvara, P., Sioufi, R., Schipper, H. M. et al.: Nitric oxidemediated erectile activity is a testosterone dependent event: a raterection model. Int J Impot Res, 7:, 209, 1995.

[0240] 8. Lekas, E., Johansson, M., Widmark, A. et al.: Decrement ofblood flow precedes the involution of the ventral prostate in the ratafter castration. Urol Res, 25:, 309, 1997.

[0241] 9. Haggstrom, S., Wikstrom, P., Bergh, A. et al.: Expression ofvascular endothelial growth factor and its receptors in the rat ventralprostate and Dunning R3327 PAP adenocarcinoma before and aftercastration. Prostate, 36:, 71, 1998.

[0242] 10. Mills, T. M., Lewis, R. W. & Stopper, V. S.: Androgenicmaintenance of inflow and veno-occlusion during erection in the rat.Biol Reprod, 59:, 1413, 1998.

[0243] 11. Mills, T. M., Stopper, V. S. & Wiedmeier, V. T.: Effects ofcastration and androgen replacement on the hemodynamics of penileerection in the rat. Biol Reprod, 51:, 234, 1994.

[0244] 12. Haggstrom, S., Lissbrant, I. F., Bergh, A. et al.:Testosterone induces vascular endothelial growth factor synthesis in theventral prostate in castrated rats. J Urol, 161:, 1620, 1999.

[0245] 13. Franck-Lissbrant, I., Haggstrom, S., Damber, J. E. et al.:Testosterone stimulates angiogenesis and vascular regrowth in theventral prostate in castrated adult rats [see comments]. Endocrinology,139:, 451, 1998.

[0246] 14. Liu X, Lin C—S, Graziottin T, Resplande J and Lue T F:Vascular endothelial growth factor promotes proliferation and migrationof cavernous smooth muscle cells. J Urol; 166: 354-360, 2001.

[0247]15. Jin K L, Mao X O, Greenberg D A.: Vascular endothelial growthfactor: direct neuroprotective effect in vitro ischemia. Proc Natl AcadSci U S A. 97:10242-7, 2000.

[0248] 16. Sandal M, Sunder F, Kanji M. Vascular endothelial growthfactor is a neurotrophic factor which stimulates axonal outgrowththrough the flk-1 receptor. Eur J Neurosci: 2000 12:4243-54, 2000.

[0249] 17. Verdonck, A., De Ridder, L., Kuhn, R. et al.: Effect oftestosterone replacement after neonatal castration on craniofacialgrowth in rats. Arch Oral Biol, 43:, 551, 1998.

[0250] 18. Parker, C. R., Jr., Ellegood, J. O. & Mahesh, V. B.: Methodsfor multiple steroid radioimmunoassay. J Steroid Biochem, 6:, 1, 1975.

[0251] 19. Maeda, Y., Ikeda, U., Ogasawara, Y. et al.: Gene transferinto vascular cells using adeno-associated virus (AAV) vectors.Cardiovasc Res, 35:, 514, 1997.

[0252] 20. Dong, J. Y., Fan, P. D. & Frizzell, R. A.: Quantitativeanalysis of the packaging capacity of recombinant adeno-associatedvirus. Hum Gene Ther, 7:, 2101, 1996.

[0253] 21. Ferrara, N., Houck, K., Jakeman, L. et al.: Molecular andbiological properties of the vascular endothelial growth factor familyof proteins. Endocr Rev, 13:, 18, 1992.

[0254] 22. Stenberg, P. E., Shuman, M. A., Levine, S. P. et al.:Redistribution of alpha-granules and their contents inthrombin-stimulated platelets. J Cell Biol, 98:, 748, 1984.

[0255] 23. Karadeniz, T., Topsakal, M., Aydogmus, A. et al.: Correlationof ultrastructural alterations in cavernous tissue with the clinicaldiagnosis vasculogenic impotence. Urol Int, 57:, 58, 1996.

[0256] 24. Baumgartner, I., Pieczek, A., Manor, 0. et al.: Constitutiveexpression of phVEGF165 after intramuscular gene transfer promotescollateral vessel development in patients with critical limb ischemia[see comments]. Circulation, 97:, 1114, 1998.

[0257] 25. Symes, J. F., Losordo, D. W., Vale, P. R. et al.: Genetherapy with vascular endothelial growth factor for inoperable coronaryartery disease. Ann Thorac Surg, 68:, 830, 1999.

[0258] 26. Mills, T. M., Dai, Y., Stopper, V. S. et al.: Androgenicmaintenance of the erectile response in the rat. Steroids, 64:, 605,1999.

[0259] 27. Hatzichristou, D. G., Saenz de Tejada, I., Kupferman, S. etal.: In vivo assessment of trabecular smooth muscle tone, itsapplication in pharmaco-cavernosometry and analysis of intracavernouspressure determinants [see comments]. J Urol, 153:, 1126, 1995.

[0260] 28. Karadeniz, T., Ariman, A., Topsakal, M. et al.: Value ofcolor Doppler sonography in the diagnosis of venous impotence. Urol Int,55:, 143, 1995.

EXAMPLE 3 The Effect of Adeno-Associated Virus-Mediated Brain-DerivedNeurotrophic Factor (BDNF) in an Animal Model for Neurogenic Impotence

[0261] Animals

[0262] Male Sprague-Dawley rats (N=34; age, 3 months; weight, 350 to 400gm.) were divided into two groups: sham (N=10) and experimental (N=24).The rats in the sham group underwent periprostatic dissection andidentification of bilateral cavernous nerves without other manipulation;those in the experimental groups underwent bilateral cavernous nervefreezing. Several minutes after surgery, half of the experimentalanimals (LacZ group, N=12) received intracavernous AAV-LacZ injection,and the remainder (BDNF group, N=12) received AAV-BDNF. Half of the ratsin each group were sacrificed at week 4 and the rest at week 8 forcollection of penile tissue. In all animals, erectile function wasassessed by electrostimulation of the cavernous nerves before sacrifice.

[0263] Surgical Procedure and Transfection Technique

[0264] Under intraperitoneal pentobarbital sodium anesthesia (50mg./kg.), each animal was placed on a heating pad to maintain its bodytemperature at 37° C. Through a lower abdominal midline incision, thearea posterolateral to the prostate was explored. The major pelvicganglia and the cavernous nerve were identified with an operatingmicroscope (Olympus, 10-40×). In the experimental group, the cavernousnerve was frozen bilaterally for 1 min. with a thermocouple used tocontrol the temperature (5 mm. diameter, Omega HH21 handheldmicroprocessor digital thermometer). (Before surgery, the thermocouplehad been placed in a 15-ml. disposable centrifuge tube filled withground dry ice and kept in a thermo-flask [Lab-Line Instruments Inc.]filled with dry ice.) The temperature of the probe at the beginning ofthe procedure was −80° C., increasing to −50° C. at 1 min. To preventdisruption of the nerve, 0.2 ml. saline was used to disengage the tip ofthe probe from the nerve before removal.

[0265] After the freezing procedure, the right side of the proximal cruswas exposed, and 0.05 ml. of either 10¹⁰ AAV-LacZ or 10¹⁰ AAV-BDNF wasinjected into 12 rats each through a tuberculin syringe with a 30Gneedle.

[0266] Preparation of AAV-BDNF

[0267] Cloning of BDNF cDNA:

[0268] We used RT-PCR to identify BDNF expression in a humanneuroblastoma cell line, SK-N-BE(2). We then used a primer pair,5′-CCCTACAGGTCGACCAGGTGA-3′ (SEQ ID NO:1) and5′-CTATACAACATGGATCCACTA-3′ (SEQ ID NO:2), to amplify the codingsequence of BDNF from SK-N-BE(2) cDNA (underlined sequences are designedXhoI and BamHI restriction sites, respectively). After digestion withXhoI and BamHI, the amplified product was cloned into pBluescript(Stratagene Inc., La Jolla, Calif.) and fully sequenced. The BDNF cDNAwas then recloned into pcDNA4, a modified version of pcDNA3 plasmid(Invitrogen, Inc., Carlsbad, Calif.) that contains the cytomegalovirus(CMV) promoter for driving the expression of BDNF in mammalian cells.

[0269] Construction of rAAV-BDNF:

[0270] The above pcDNA4BDNF was digested with SalI to release theexpression cassette containing cytomegalovirus immediate-early (CMVie)promoter, BDNF gene and bovine growth hormone (BGH) poly-A signal. Thisexpression cassette was inserted into an AAV vector, pAV53, resulting inthe construction of rAAV-BDNF.

[0271] Virus Production and Titration:

[0272] rAAV-BDNF was produced by a three-plasmid co-transfection method.Twenty 15-cm plates of 293 cells (50 to 60% confluent) were maintainedin Dulbecco modified Eagle medium (DMEM, Gibco) supplemented with 10%fetal bovine serum (Hyclone) and 25 mM. HEPES and co-transfected by thecalcium phosphate method with a total of 45 μg. DNA, 15 pg. of AAV-BDNFvector, and 15 μg. each of pLHP19 (AAV helper plasmid) and pLadeno5(adenovirus helper plasmid), kindly provided by AVIGEN. Six hours aftertransfection, the medium was replaced with fresh DMEM containing 1%fetal bovine serum. The cells were harvested at 48 h posttransfection bycentrifugation (1000 g for 10 min.), and the cell pellets werere-suspended in 0.1 M. Tris-HCL, 0.15 M. NaCl solution (pH 8.0) andsubjected to four cycles of freeze-thaw and removal of cell debris.Large-scale rAAV CsCl purification was carried out as describedpreviously.¹² AAV-BDNF vector titer was determined by quantitative dotblot hybridization of DNase-treated stocks. The AAV vector titer used inthe experiment refers to the particle number of AAV vector genomes inthe sample as determined by the quantitative dot blot assay.

[0273] Functional Evaluation and Tissue Procurement

[0274] At weeks 4 and 8 postoperatively, rats in each group werere-explored for direct electrostimulation of the cavernous nerves beforetissue collection. The skin overlying the penis was incised and theischiocavernous muscle was partly removed to expose both penile crura. A23G butterfly needle connected to PE-50 tubing was inserted in the rightcrus for pressure measurement. Electrostimulation was performed with adelicate stainless-steel bipolar hook electrode attached to amulti-jointed clamp. (Each pole was 0.2 mm. in diameter; the two poleswere separated by 1 mm.) Short wave pulses were generated by a Macintoshcomputer with a custom-built constant current amplifier. Stimulusparameters were 1.5 mA., frequency 20 Hz., pulse width 0.2 m sec.,duration 50 sec. Each cavernous nerve was stimulated and intracavernouspressures were measured and recorded with a Macintosh computerprogrammed with LabVIEW 4.0 software (National Instruments, Austin,Tex.). The pressure for each animal was determined by the mean of bothsides.

[0275] NADPH Diaphorase Staining

[0276] After sacrifice, samples of major pelvic ganglia and peniletissue were fixed for 3 hours in a cold, freshly prepared, solution of2% formaldehyde, 0.002% picric acid in 0.1 M. phosphate buffer, pH 8.0.Tissues were cryoprotected for 24 hours in cold 30% sucrose in 0.1 M.phosphate buffer, pH 8.0. They were then embedded in O.C.T. compound(Tissue-Tek, Miles Laboratory), frozen in liquid nitrogen, and stored at−70° C. Cryostat tissue sections were cut at 10 μm., adhered to chargedslides, air-dried for 5 min., and hydrated for 5 min. with 0.1 M. PO₄,pH 8.0. Sections were incubated with 0.1 mM. NADPH, 0.2 mM. nitrobluetetrazolium, 0.2% Triton X-100 in 0.1 M. PO₄, pH 8.0, for 60 min. atroom temperature. The reaction was terminated by washing in buffer.Slides were then coverslipped with buffered glycerin as the mountingmedium.¹³

[0277] The presence of NADPH diaphorase-positive nerves was evidenced asa blue stain in the major pelvic ganglia, dorsal nerves and cavernoustissue, and the staining pattern was assessed by counting the number ofpositive neurons in 4 random fields (magnification 400×). The percentageof darkly and lightly stained cells in the major pelvic ganglia wascalculated by dividing the number of these cells by the total number ofpositive cells.

[0278] Nitric Oxide Synthase Antibody Staining

[0279] Tissue fixation was the same as with NADPH-diaphorase specimens.After freezing, 10 μm. cryostat tissue sections were adhered to chargedslides, air-dried, and hydrated for 5 min. with 0.05M. sodium phosphatebuffer (PBS, pH 7.4). Sections were treated with hydrogenperoxide/methanol to quench endogenous peroxidase activity. Afterrinsing with water, sections were washed twice in PBS for 5 min.followed by 30 min. of room-temperature incubation with 3% horseserum/PBS/0.3% triton X-100. After draining solution from sections,tissues were incubated for 60 min. at room temperature with mousemonoclonal anti-nNOS (Transduction Laboratories, Lexington, Ky.) at a1:500 dilution. After washing for 5 min. with PBS/TX and then for 5 min.twice with PBS alone, sections were immunostained with theavidin-biotin-peroxidase method (Elite ABC, Vector Labs, BurlingameCalif.), with diaminobenzidine as the chromagen, followed bycounterstaining with hematoxylin.

[0280] Statistical Analysis: The nonparametric Mann-Whitney U and ANOVAtests with Statview 4.02 software were used to compare results. Valueswere considered significant at p<0.05.

Results

[0281] Functional Studies

[0282] In both the LacZ and BDNF groups, maximal intracavernous pressurein response to bilateral cavernous nerve electrostimulation was lessthan in the sham group. However, the pressure in the BDNF group wassignificantly higher than in the LacZ group at both 4 and 8 weeks (FIG.1 and Table 6). TABLE 6 Maximal intracavernous Pressure In Response toElectrostimulation 4 and 8 Weeks After Bilateral Cavernous NerveFreezing Time Sham Operation LacZ BDNF (Weeks) (n = 10) (n = 12) (n =12) 4   105 ± 10.5* 28.4 ± 5.5  585 ± 11.7† 8 115.5 ± 7.7 37.7 ± 7.961.3 ± 12.5†

[0283] NADPH Diaphorase Staining

[0284] Dorsal Nerve:

[0285] At week 4, the LacZ group showed significantly fewer NADPHdiaphorase-positive nerve fibers than did the BDNF group. At week 8, thenumber had increased in both groups, but there were still significantlyfewer in the LacZ group When compared with the sham group, bothexperimental groups showed fewer positive nerve fibers (Table 7). TABLE7 NADPH Diaphorase-positive Nerve Fibers In the Dorsal Nerve andCavernous Tissue Time Sham Operation LacZ BDNF (Weeks) (N = 10) (N = 12)(N = 12) Dorsal Nerve 4 Weeks 138.5 ± 10.5* 45.7 ± 5.8 65.5 ± 15.5^(†) 8Weeks 140.2 ± 9.8 55.6 ± 8.4 86.4 ± 12.2^(†) Cavernous Tissue 4 Weeks  103 ± 9.8 25.5 ± 3.6 45.5 ± 10.5^(†) 8 Weeks 110.2 ± 10.5 35.6 ± 10.466.1 ± 15.2^(†)

[0286] Intracavernous Nerves:

[0287] Histologic evaluation showed significantly fewer NADPHdiaphorase-positive nerve fibers in the trabecular smooth muscle of theLacZ group than in the BDNF group at both 4 and 8 weeks. When comparedwith the sham group, both experimental groups had significantly fewer(Table 7).

[0288] Major Pelvic Ganglia:

[0289] At week 4, most of the neurons in the major pelvic ganglia in theLacZ group exhibited a lighter staining pattern than that seen in theBDNF group. In addition, at 8 weeks most of these LacZ neurons hadirregular cell contour and multiple vacuoles in the cytoplasm. Thepercentage of darkly stained cells in the BDNF group at both time pointswas significantly higher than in the LacZ group, and their appearance(i.e. smooth contour and very few vacuoles in the cytoplasm) was similarto that in the sham group. In both experimental groups, the percentageof darkly stained cells was significantly less than in the sham group(Table 8). TABLE 8 NADPH Diaphorase-positive Neurons in the Major PelvicGanglion % % Dark Light Dark Light Time Group Stain Stain Total StainStain 4 Sham 102 ± 21.7 ± 123.5 ± 82.2 ± 17.3 ± Weeks 0.5* 3.5 4.5^(†)5.3 6.8 LacZ 32.4 ± 52.5 ± 85 ± 20.1 38.5 ± 60.7 ± 15.2 20.4 17.2 17.2BDNF 61.8 ± 33.8 ± 9 95.7 ± 62.1 ± 38.6 ± 23.7 22.4 13.1^(‡) 11.8 8 Sham101 ± 25.5 ± 136.7 ± 80.7 ± 19.2 ± Weeks 12.3 5.5 11.3^(†) 9.8 4.5 LacZ45 ± 12.7 53.3 ± 98.5 ± 45.8 ± 54.1 ± 9.5 14.3 0.8 0.8 BDNF 69.4 ± 38.4± 14 107.8 ± 64.3 ± 35.6 ± 21.1 23.58 10.7^(‡) 10.7

[0290] nNOS Immunostaining

[0291] Immunostaining of penile tissue and the major pelvic ganglia fornNOS revealed positive staining in the same nerve fibers and neurons aswith NADPH diaphorase. The neurons of the major pelvic ganglia of theLacZ group showed lighter staining patterns and many more vacuoles inthe cytoplasm than did the neurons in the BDNF and sham groups.

Discussion

[0292] The aim of the present study was to investigate the feasibilityof using AAV-BDNF gene transfer to facilitate recovery of potency afterbilateral cavernous nerve injury. Our past studies have led us tobelieve that bilateral cavernous nerve freezing in the rat is a suitablemodel for such injury because the neural sheath is preserved, as it isin patients undergoing nerve-sparing prostatectomy or cryoablation. Inaddition, the course and extent of functional recovery have been welldocumented in this model.¹⁴ We used maximal intracavernous pressure inresponse to cavernous nerve electrostimulation to assess recovery oferectile function. Although apomorphine-induced erection may be morephysiologic, we do not believe it can reliably differentiate partialfrom full erection in rats with cavernous nerve injury.

[0293] In this study, the number of nNOS-containing neurons in the majorpelvic ganglia of both experimental groups was less than in the shamgroup. However, the percentage of darkly stained neurons wassignificantly greater in the BDNF group than in the LacZ group at both 4and 8 weeks. Moreover, most neurons of the BDNF and sham groups did notshow the cytoplasmic vacuoles and irregular cell contour seen in theLacZ group. These findings suggest that the production of BDNF proteinin penile tissue can be retrogradely transported to the major pelvicganglia to prevent neuronal damage and preserve nNOS enzymes in theneurons. This in turn facilitates the recovery of erectile function, asevidenced by the more numerous nNOS-positive nerve fibers in theerectile tissue and higher intracavernous pressure in the BDNF group.

[0294] A previous study has shown a significantly increased survival ofmotoneurons 1 week after axotomy in animals pretreated withadenovirus-encoding BDNF or glial cell line-derived neurotrophic factor(GDNF).¹⁷ However, because of the disadvantages of the adenovirus, weused adeno-associated virus (AAV), a unique member of the non-enveloped,single-stranded-DNA Parvovirus that possesses several properties thatdistinguish it from other gene-transfer vectors. Its advantages includestable and efficient integration of viral DNA into the host genome,¹⁸lack of associated human disease,¹⁹ broad host range, ability to infectgrowth-arrested cells,²⁰ and ability to carry non-viral regulatorysequences without interference from the viral genome.¹⁸ In addition, nosuperinfection inhibition is associated with AAV vectors.¹⁸ The infectedcells are spread several millimeters around the needle tract. AAV isable to infect axon terminals and is retrogradely transported. Injectionof AAV vector expressing LacZ into several brain regions has shown thepresence of transgene expression as early as 24 hours,²² lasting (atsignificantly decreased levels) as long as 6 months.

[0295] The penis is a convenient organ for gene therapy because of itsexternal location and slow circulation in the flaccid state. Inaddition, its sinusoidal structure and the gap junctions between smoothmuscles ensure wide distribution of injected vectors. To our knowledge,this is the first demonstration of gene therapy with AAV-BDNF used tofacilitate the recovery of nNOS-containing nerves and neurons andconsequent erectile function. Because our previous studies have shownthat 3 to 6 months may be required for more complete regeneration ofcavernous nerves and erectile function in both unilateral resection andunilateral freezing models,^(3,16) we are presently conducting a furtherstudy to examine the effects of higher AAV-BDNF titer and longerfollow-up in this model.

Conclusion

[0296] Our results showed that intracavernous injection of AAV-BDNFafter freezing of bilateral cavernous nerves had the followingeffects: 1) facilitated the recovery of erectile function, 2) enhancedthe regeneration of the intracavernous and dorsal nerves, and 3)prevented neuronal degeneration in the major pelvic ganglia. If furtherstudies confirm its effectiveness and safety, intracavernous injectionof neurotrophins or other growth factors has the potential to be acurative therapy for neurogenic erectile dysfunction after cryoablationor radical pelvic surgery. Bakircioglu ME, et al., J Urol. 165:2103-9(2001).

REFERENCES

[0297] 1. Walsh, P. C., and Mostwin, J. L. Radical prostatectomy andcystoprostatectomy with preservation of potency. Results using a newnerve-sparing technique. Br. J. Urol., 56: 694, 1984.

[0298] 2. Paick, J. S., Donatucci, C. F., and Lue, T. F. Anatomy ofcavernous nerves distal to prostate: microdissection study in adult malecadavers. Urology, 42: 145, 1993.

[0299] 3. Carrier, S., Zvara, P., Nunes, L., Kour, N. W., Rehman, J.,and Lue, T. F. Regeneration of nitric oxide synthase-containing nervesafter cavernous nerve neurotomy in the rat. J. Urol. 153: 1722, 1995.

[0300] 4. Jung, G. W., Spencer, E. M., and Lue, T. F. Growth hormoneenhances regeneration of nitric oxide synthase-containing penile nervesafter cavernous nerve neurotomy in rats. J. Urol. 160: 1899, 1998.

[0301] 5. Leibrock, J., Lottspeich, F., Hohn, A., Hofer, M., Hengerer,B., Masiakowski, P., Thoenen, H., and Barde, Y. A. Molecular cloning andexpression of brain-derived neurotrophic factor. Nature. 341: 149, 1989.

[0302] 6. Ide, C.: Peripheral nerve regeneration. Neurosci. Res. 25:101, 1996. 7. DiStefano, P. S., Friedman, B., Radziejewski, C.,Alexander, C., Boland, P., Schick, C. M., Lindsay, R. M., and Wiegand,S. J. The neurotrophins BDNF, NT-3, and NGF display distinct patterns ofretrograde axonal transport in peripheral and central neurons. Neuron.8: 983, 1992.

[0303] 8. Oppenheim, R. W., Yin, Q. W., Prevette, D., and Yan, Q.Brain-derived neurotrophic factor rescues developing avian motoneuronsfrom cell death. Nature. 360: 755, 1992.

[0304] 9. Yan, Q., Elliott, J., and Snider, W. D. Brain-derivedneurotrophic factor rescues spinal motor neurons from axotomy-inducedcell death. Nature. 360: 753, 1992.

[0305] 10. Meyer, M., Matsuoka, I., Wetmore, C., Olson, L., and Thoenen,H. Enhanced synthesis of brain-derived neurotrophic factor in thelesioned peripheral nerve: different mechanisms are responsible for theregulation of BDNF and NGF mRNA. J. Cell Biol. 119: 45, 1992.

[0306] 11. Nonomura, T., Nishio, C., Lindsay, R. M., and Hatanaka, H.Cultured basal forebrain cholinergic neurons from postnatal rats showboth overlapping and non-overlapping responses to the neurotrophins.Brain Res. 683: 129, 1995.

[0307] 12. Matsushita, T., Elliger, S., Elliger, C., Podsakoff, G.,Villarreal, L., Kurtzman, G. J., Iwaki, Y., and Colosi, P.Adeno-associated virus vectors can be efficiently produced withouthelper virus. Gene Ther. 5: 938, 1998.

[0308] 13. Alm, P., Larsson, B., Ekblad, E., Sundler, F., and Andersson,K. E. Immunohistochemical localization of peripheral nitric oxidesynthase-containing nerves using antibodies raised against synthesizedC- and N-terminal fragments of a cloned enzyme from rat brain. ActaPhysiol. Scand. 148: 421, 1993.

[0309] 14. Korsching, S. The neurotrophic factor concept: areexamination. J. Neurosci. 13: 2739, 1993.

[0310] 15. Lewin, G. R. and Barde, Y. A. Physiology of theneurotrophins. Annu. Rev. Neurosci. 19: 289, 1996.

[0311] 16. El-Sakka, A. I., Hassan, M. U., Selph, C., Perinchery, G.,Dahiya, R., and Luc, T. F. Effect of cavernous nerve freezing on proteinand gene expression of nitric oxide synthase in the rat penis and pelvicganglia. J. Urol. 160: 2245, 1998.

[0312] 17. Gimenez y Ribotta, M., Revah, F., Pradier, L., Loquet, I.,Mallet, J., and Privat, A. Prevention of motoneuron death byadenovirus-mediated neurotrophic factors. J. Neurosci. Res. 48: 281,1997.

[0313] 18. McLaughlin, S. K., Collis, P., Hermonat, P. L., and Muzyczka,N. Adeno-associated virus general transduction vectors: analysis ofproviral structures. J. Virol. 62: 1963, 1988.

[0314] 19. Berns, K. I., Cheung, A., Ostrove, J., and Lewis, M.:Adeno-associated virus latent infection. In: B. W. J. Mahy, A. C.Minson, and G. K. Darby (eds.), Virus Persistence. Cambridge, UK:Cambridge University Press, 1982.

[0315] 20. Podsakoff, G., Wong, K. K., Jr., and Chatterjee, S. Efficientgene transfer into nondividing cells by adeno-associated virus-basedvectors. J. Virol. 68: 5656, 1994.

[0316] 21. Miller, J. L., Walsh, C. E., Ney, P. A., Samulski, R. J., andNienhuis, A. W. Single-copy transduction and expression of humangamma-globin in K562 erythroleukemia cells using recombinantadeno-associated virus vectors: the effect of mutations in NF-E2 andGATA-1 binding motifs within the hypersensitivity site 2 enhancer[published erratum appears in Blood 1995 Feb 1;85(3):862]. Blood. 82:1900, 1993.

[0317] 22. During, M. J. and Leone, P. Adeno-associated virus vectorsfor gene therapy of neurodegenerative disorders. Clin. Neurosci. 3: 292,1995.

EXAMPLE 4 Vascular Endothelial Growth Factor Promotes Proliferation andMigration of Cavernous Smooth Muscle Cells

[0318] Animals

[0319] Male Wistar rats were obtained from Charles River Laboratories(Wilmington, Mass.). Young rats were 1, 2, 3, 4, 6, 11, and 16 weeks ofage. Old rats were 28 months of age.

[0320] Regents

[0321] All chemicals were from Sigma-Aldrich Co. (St. Louis, Mo.) unlessnoted otherwise. Recombinant human VEGF₁₆₅ was from CalbiochemBiosciences Inc. (La Jolla, Calif.). Fetal bovine serum (FBS) andTrypsin-EDTA were from Life Technologies, Inc. (Grand Island, N.Y.). Allother cell culture regents were obtained from Cell Culture Facility,University of California, San Francisco.

[0322] Cell Culture

[0323] Each primary culture of CSMC was prepared from the corporacavernosa of 2-3 rats by the following procedure. The penis was clearedof the urethra, blood vessels, fat and connective tissue. The remainingsmooth muscle tissue was washed 3 times in sterile PBS(phosphate-buffered saline) and cut into 2-3 mm³ segments. The segmentswere placed evenly onto a 100-mm cell culture dish (Falcon-BectonDickinson Labware, Franklin Lakes, N.J.) inside a cell culture hood.Approximately 10 min later, 10 ml of Dulbecco's Modified Eagle Medium(DMEM) containing penicillin (100 units/ml), streptomycin (100 μg/ml),and 10% FBS was carefully pipetted into the dish. The dish was then keptundisturbed in a humidified 37° C. incubator with 5% CO₂. Five dayslater, tissue segments that have detached from the dish were removed,and the medium was replaced with fresh medium. Another 5 days later, alltissue segments were removed and the medium was again replaced withfresh medium. When small islands of cells were noticeable, they weretrypsinized and transferred to a fresh culture dish. Expansion of eachcell strain was continued with change of medium every 3 days andpassages (trypsinization and seeding) approximately every 10 days. Allcells used in the following experiments were from passages 4 through 10.Primary aorta SMC cultures were prepared similarly with aortas isolatedfrom 16-week-old male rats. All cell cultures were confirmed for theirsmooth muscle identity by an indirect immunofluorescence staining withan anti-smooth muscle myosin heavy chain antibody (Sigma-Aldrich Co. St.Louis, Mo.).

[0324] Quantification of VEGF

[0325] Each CSMC from rats of different ages was seeded at 4×10⁵ cellsper well in 3 ml of DMEM with 10% FBS in 6-well culture plates.Seventy-two hr later, the medium was removed for quantification of VEGFand the cells were trypsinized for the determination of cell number. Forquantifying VEGF in the medium, the Mouse VEGF Immunoassay Kit (R&DSystems, Minneapolis, Minn.), which reacts with rat VEGF but not withbovine VEGF, was used. All assays were performed in duplicate in eachexperiment and all data presented in the Results section are the averageof three independent experiments.

[0326] Proliferation Assay

[0327] Cell proliferation assays were performed with the CellTiter-96kit from Promega Inc. (Madison, Wis.). Each SMC strain fromdifferent-aged rats was assayed in one flat-bottom 96-well cell cultureplate. The plate was divided into 12 rows that contained VEGF atconcentrations from 0 to 100 ng per ml of serum-free DMEM (supplementedwith 0.1% BSA). Each well in the same row received 50 μl of the mediumcontaining the same concentration of VEGF. Thereafter, SMC that weregrown to 70% confluence were rinsed twice with PBS, trypsinized, andresuspended in serum-free DMEM (supplemented with 0.1% BSA) at 100,000cell per ml. Aliquots of 50 μl of the cell suspension were thentransferred to the 96-well plate so that each well contained 5,000 cellsin a final volume of 100 μl. The plate was incubated in a 37° C.,humidified incubator with 5% CO₂. Three days later, 20 μl of CellTiter96®AQueous One Solution Reagent was added to each well. After 4 hr offurther incubation at 37° C. in the humidified, 5% CO₂ incubator, colordevelopment, which reflects cell numbers, was recorded with a platereader (Molecular Devices Corp., Sunnyvale, Calif.) at 490-nmabsorbance. For proliferation assays concerning concentrations of FBS inthe growth medium, the assay procedure was the same except thatdifferent amounts of FBS, instead of VEGF, were added to the medium. Allassays were performed in duplicate in each experiment and all datapresented in the Results section are the average of three independentexperiments.

[0328] Migration Assay

[0329] Cell migration assays were performed in 6.5-mm Transwell chambersof Coming Costar Corporation (Cambridge, Mass.). The Transwell inserts(upper chambers) were bathed in a solution containing 13.4 μg/mlfibronectin in PBS at 37° C. for 1 hr and allowed to air-dry. The driedupper chambers were then placed in the lower chambers, each of whichcontained 700 μl of serum-free DMEM supplemented with 0.1% BSA and 0 to500 ng/ml of VEGF. SMC that had been grown to 70% confluence werefurther conditioned in serum-free DMEM (containing 0.1% BSA) for 2 hrand then trypsinized. The trypsinized cells were washed in PBS andresuspended at a concentration of 80,000 cells per ml in serum-free DMEM(containing 0.1% BSA). One hundred μl of the cell suspension was thenadded to each upper chamber. After 4 hr of incubation at 37° C., allliquid in the upper and lower chambers was removed by aspiration. Themembranes in the upper chambers were subsequently fixed in 1% bufferedformalin for 5 min and stained with 2% crystal violet. Non-migratorycells on the upper side of the membranes were scraped off with cottonswabs. Well locations were marked on the membranes and total cells perwell were counted visually in a masked fashion. Well locations were thencorrelated with the concentrations of VEGF in the wells. All assays wereperformed in duplicate in each experiment and all data presented in theResults section are the average of three independent experiments.

[0330] RNA Preparation

[0331] Cultured cells and rat tissues were homogenized in Tri-ReagentRNA extraction solution (Molecular Research Center, Cincinnati, Ohio).Following the recommended procedure by the supplier, RNAs were furthertreated with DNase I to remove traces of contaminating DNA. Quantity andintegrity of RNAs were examined by spectrophotometry and agarose gelelectrophoresis, respectively. Human heart RNAs were purchased fromClontech Laboratories, Inc. (Palo Alto, Calif.).

[0332] RT-PCR Analysis

[0333] RT-PCR (reverse transcription-polymerase chain reaction) wasperformed in an RT step and a PCR step. In the RT step, the cellularmRNAs were reverse-transcribed into a “library” of complementary DNAs(cDNAs). This cDNA library was then used for the analysis of variousgenes in the PCR step. The RT procedure was performed with theSuperScript reverse transcriptase (Life Technologies, Inc.,Gaithersburg, Md.) and its accompanying reagents. Briefly, 2.5 μg ofeach tissue RNA was annealed to 0.4 μg of oligo-dT primer in a 12 μlvolume. Four μl of 5× buffer, 2 μl of 0.1 M DTT, 1 μl of 10 mM dNTP, and1 μl of SuperScript reverse transcriptase were then added to bring thefinal reaction volume to 20 μl. After one hour of incubation at 42° C.,the RT mixture was incubated at 70° C. for 10 min to inactivate thereverse transcriptase. Eighty μl of TE buffer was then added to make a5× diluted library. A portion of this library was further diluted tovarious concentrations (up to 100× dilution). One μl of each dilutionwas then used in a 10 μl PCR to identify the optimal input within thelinear amplification range. In addition to the 1 μl diluted library, thePCR mixture consisted of 10 ng of each of a primer pair and reagentssupplied with the Taq polymerase (Life Technologies, Inc., Gaithersburg,Md.). PCR was performed in the DNA Engine thermocycler (MJ Research,Inc., Watertown, Mass.) under calculated temperature control. Thecycling program was set for 35 cycles of 94° C., 5 sec; 55° C., 5 sec;72° C., 10 sec, followed by one cycle of 72° C., 5 min. The PCR productswere electrophoresed in 1.5% agarose gels in the presence of ethidiumbromide, visualized by UV fluorescence, and recorded by a digital cameraconnected to a computer. TABLE 9 Oligonucleotide primers Size of PrimerPCR Gene name Sequence product β-Actin Actin-s5′-TCTACAATGAGCTGCGTGTG-3′ 368 bp (SEQ ID NO:3) Actin-a3′-AATGTCACGCACGATTTCCC-5′ (SEQ ID NO:4) VEGFR-1 VEGFR-1s5′-ATGCTGGATTGCTGGCACA-3′ 323 bp (SEQ ID NO:5) VEGFR-1a3′-TCAAACATGGAGGTGGCATT-5′ (SEQ ID NO:6) VEGFR-2 VEGFR-2s5′-GCCTTTGGCCAAGTGATTGA-3′ 479 bp (SEQ ID NO:7) VEGFR-2a3′-TCCAAGGTCAGGAAGTCCTT-5′ (SEQ ID NO:8)

[0334] Oligonucleotide Primers

[0335] Primer pairs for RT-PCR analysis of VEGFR-1, VEGFR-2 and β-actingenes are listed in Table 9. They were designed to recognize therespective mRNAs in both humans and rats.

[0336] Western Blot Analysis

[0337] Cultured cells were lysed in a buffer containing 1% IGEPALCA-630, 0.5% sodium deoxycholate, 0.1% SDS, 10 μg/ml aprotinin, 10 μg/mlleupeptin, and 1× PBS. Cell lysates containing indicated amounts ofprotein were electrophoresed in 7.5% SDS-PAGE and then transferred toPVDF membrane. The membrane was stained with Ponceau S to verify theintegrity of the transferred proteins and to monitor the unbiasedtransfer of all protein samples. Detection of VEGFR-1 protein on themembrane was performed with the ECL kit (Amersham Life Sciences Inc.,Arlington Heights, Ill.) using an anti-VEGFR-1 rabbit serum from SantaCruz Biotech, Inc. (Santa Cruz, Calif.).

Results

[0338] Growth Rates of CSMC from Different-Aged Rats

[0339] Like most other cultured cells, our rat CSMC were maintained in amedium supplemented with 10% FBS. However, because certain experimentsmay require the use of media containing lower concentrations of FBS, wewished to know the differences between different age groups in theirgrowth rates under different concentrations of FBS. All cells,regardless of the ages of rats from which they were derived, grew atincreasing rates with increasing FBS concentrations. At lowerconcentrations of FBS (0 and 2.5%), the growth rates showed littledifferences between different age groups. However, at higherconcentrations of FBS (5 and 10%), differences in growth rates becamemore pronounced. In particular, cells from 4-week-old rats seemed torespond best to higher concentrations of FBS and cells from 28-month-oldrats least well.

[0340] VEGF Secretion by CSMC from Different-Aged Rats

[0341] VSMC have been shown to be the principal source of secreted VEGFin the vascular (aorta) system. Pueyo, et al., Exp. Cell Res., 238: 354(1998). We therefore examined our rat CSMC for their ability to produceVEGF. We chose 72 hr after seeding the cells as the point of time toassay for the secreted VEGF. Because CSMC from different age groups grewat different rates, we also determined the cell numbers for each of thetested cells. When the concentrations of the secreted VEGF were adjustedfor the numbers of cells at the time of assay (FIG. 2A), it is apparentthat, within the young rat groups (ages 1 to 16 weeks), CSMC from morematured rats secreted more VEGF than CSMC from less matured rats (FIG.2C). However, CSMC from old rats (28 months old) produced similaramounts of VEGF as those from the very young rats (1 and 2 weeks old).

[0342] Effects of VEGF on Cell Growth

[0343] It has been reported that VEGF did not stimulate growth of VSMC.Grosskreutz, et al., Microvasc. Res., 58: 128 (1999). This observationwas confirmed with VSMC from 16-weeks-old rats (FIG. 3A). However, CSMCfrom all ages of rats responded to VEGF in the form of cellproliferation. Their growth rates increased with increasingconcentrations of VEGF up to 12.5 ng/ml, and after which, the cellgrowth rate started to decline with increasing concentrations of VEGF(FIGS. 3B-I). When compared at the optimal dosage (12.5 ng/ml) of VEGF,all cells from young rats (1 to 16 weeks old) outgrew cells from the oldrats (28 months) and the peak growth occurred with cells from11-weeks-old rats (FIG. 3J).

[0344] Effects of VEGF on Cell Motility

[0345] It has been reported that VEGF stimulated migration of VSMC.Grosskreutz, et al., Microvasc. Res., 58: 128 (1999). This observationwas confirmed with VSMC from 16-weeks-old rats (FIG. 4A). Similarly,VEGF stimulated migration of CSMC from both young and old rats in adose-dependent manner up to the 10 ng/ml point (except the 1-week-old,which peaked at 1 ng/ml). At higher concentrations (100 and 500 ng/ml)of VEGF, the mobility of all tested cells started to decline (FIGS.4B-I). When compared at the optimal dosage (10 ng/ml) of VEGF, all cellsfrom young rats (1 to 16 weeks old) out-migrated cells from the old rats(28 months) and the peak migration rate occurred with cells from4-weeks-old rats (FIG. 4J).

[0346] Identification of VEGFR-1 and VEGFR-2 mRNA Expression

[0347] The above cell proliferation and migration assay resultssuggested the presence of functional VEGF receptors in CSMC. To verifythis, we use RT-PCR to examine the CSMC for the expression of VEGFR-1and VEGFR-2 mRNAs. As shown in FIG. 5A, CSMC of different-aged ratsexpressed VEGFR-1 mRNA at different levels, being very low for the youngones (1 to 3 weeks of age) and the very old (28 months of age) and highfor the adolescent (4 and 6 weeks of age) and the matured (11 and 16weeks of age). This pattern of VEGFR-1 mRNA expression is similar tothat of VEGF-induced cell proliferation (FIG. 3).

[0348] On the other hand, CSMC, regardless of the ages of rats fromwhich they were derived, did not express VEGFR-2 (Lanes 1 to 8, FIG.5B). This negative result could not have been due to improper RT-PCRconditions or improper primer design because rat heart, aorta, and penisall produced positive results under the same experimental conditions(lanes 10, 11, and 12, FIG. 5B). The positive VEGFR-2 expression inheart, aorta, and penis was most likely derived from vascularendothelial cells that were included in the preparation of these tissueRNAs. It should be pointed out that our rat VSMC were also negative forVEGFR-2 expression (Lane 9, FIG. 5B). This is in agreement with aprevious study which reported that SMC of rat carotid arteries expressVEGFR-1 but not VEGFR-2. Couper, et al., Circ. Res., 81: 932 (1997).

[0349] Identification of VEGFR-1 Protein Expression

[0350] To ascertain that VEGFR-1 protein was indeed expressed in CSMC,we performed immunoblotting experiments using a VEGFR-1-specificantibody. As shown in FIG. 6, VEGFR-1 was detected in CSMC from rats ofall ages, and the levels of its expression was very similar to thoseseen in the results of RT-PCR experiments (FIG. 5A). Therefore, both theexpression of VEGFR-1 mRNA and VEGFR-1 protein correlated well with theVEGF-regulated growth rate of CSMC.

Discussion

[0351] The erectile function of the penis is that of a vascular organ.Like those of other vascular organs, the development and growth of thepenile vasculature are expected to be governed by angiogenic growthfactors such as VEGF. Surprisingly, reports concerning VEGF expressionin the penis have been scant. In two separate studies, Burchardt et al.reported the identification of novel VEGF splice variants in the penis.Burchardt et al., Biol. Reprod., 60: 398 (1999), Burchardt, et al.,IUBMB Life, 48: 405 (1999). In another study concerning the expressionof various growth factors in the penis, Jung et al. reported theidentification of VEGF₁₈₉ mRNA in the penis. Jung, et al., Int. J.Impotence Res., 11: 247 (1999). Our ischemia rat model clearlydemonstrated the beneficial effects of VEGF on the restoration of theerectile function following surgical procedures that restricted bloodsupply to the penis.

[0352] We have conducted several experiments (RT-PCR, western blots,immunohistochemical stainings) on the expression of various VEGF formsand their receptors in the penis (unpublished). In trying to interpretthe results of those experiments, we were confronted with the questionwhich cell types (smooth muscle, endothelium, nerve, etc.) were thesource of a positive gene expression. By using cultures of a single celltype, we were able to show in the present study that CSMC expressed VEGFand VEGFR-1 but not VEGFR-2. We also showed that both the secretion ofVEGF and the expression of VEGFR-1 increased with the age of young rats(from 1 to 16 weeks of age) but declined in very old rats (28 months ofage).

[0353] We are aware of only three previously published reports thatstudied the effects of VEGF on SMC or expression of VEGF receptors inSMC. First, Brown et al. showed that cultured human uterine SMCexpressed both VEGFR-1 and VEGFR-2 and responded to VEGF stimulation inthe form of cell proliferation. Brown, et al., Lab. Invest., 76: 245(1997). These authors also showed that human colon SMC did not expressVEGF receptors and did not respond to VEGF stimulation. Secondly, Couperet al. observed high levels of VEGFR-1, but no VEGFR-2, expression inSMC of rat carotid arteries following balloon injury. Couper, et al.,Circ. Res., 81: 932 (1997). Thirdly, Grosskreutz et al. showed thatcultured bovine aorta SMC expressed both VEGFR-1 and VEGFR-2 andresponded to VEGF stimulation in the form of cell migration (but notcell proliferation). Grosskreutz, et al., Microvasc. Res., 58: 128(1999). In the present study, we used rat aorta SMC for comparison withrat CSMC. We found that, like bovine aorta SMC, rat aorta SMC respondedto VEGF stimulation in the form of cell migration but not cellproliferation. However, we could only identify VEGFR-1 but not VEGFR-2expression in rat aorta SMC. Whether the discrepancy regarding VEGFR-2expression is due to species difference (bovine verses rat) or otherfactors (age) needs to be clarified in future studies.

[0354] The significance of the proliferative and migratory responses ofCSMC toward VEGF is not known. Brown et al. speculated that alterationsin expression of VEGF or VEGF receptors may play a role in thepathogenesis of smooth muscle tumors in the uterus. Brown, et al., Lab.Invest., 76: 245 (1997). Grosskreutz et al. proposed that VEGF mighthave a chemoattractant role in the recruitment of smooth muscle cellsduring the formation of a blood vessel wall. Grosskreutz, et al.,Microvasc. Res., 58: 128 (1999). Because the proliferative effect ofVEGF occurred mainly with CSMC of young adult rats (11 weeks of age), itis possible that VEGF at least play a role in maintaining a healthypopulation of CSMC. As for the migratory effect of VEGF, which occurredmainly with even younger rats (4 weeks of age), we propose that VEGFmight play a role in recruiting and/or locating CSMC to the proper sitesin the cavernous spaces during adolescence.

[0355] In conclusion, we believe our present study has made thefollowing novel observations: (1) CSMC secreted VEGF, (2) CSMC expresseda VEGF receptor, (3) CSMC exhibited migratory and proliferativeresponses to VEGF, and (4) CSMC from different-aged rats expresseddifferent levels of VEGF and VEGFR-1 and responded to VEGF at differentrates. Liu, X., et al., J. Urol 166:354-360 (2001).

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[0362] 7. Yamada, Y., Nezu, J., Shimane, M. and Hirata, Y. (1997)Genomics 42(3), 483-488.

[0363] 8. Anthony, F. W., Wheeler, T., Elcock, C. L., Pickett, M. andThomas, E. J. (1994) Placenta 15(5), 557-561.

[0364] 9. Neufeld, G., Cohen, T., Gengrinovitch, S. and Poltorak, Z.(1999) FASEB J. 13(1), 9-22.

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[0366] 11. Jingjing, L., Xue, Y., Agarwal, N. and Roque, R. S. (1999)Invest. Ophthal. Vis. Sci., 40(3), 752-759.

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EXAMPLE 5 The Effect of a Vascular Endothelial Growth Factor (VEGF) andAdeno-Associated Brain Derived Neurotrophic Factor (AAV-BDNF) for theTreatment of Erectile Dysfunction Induced by Hypercholesterolemia in aRat Model

[0381] Twenty-one Sprague-Dawley rats were used. All rats were fed a2.5% cholesterol diet with added lard starting at 2 weeks of age for 6months. The rats were divided into three groups. All groups after twomonths of a high cholesterol diet underwent serum evaluation ofcholesterol and intracavemous injection of saline or treatment. Group 1,the control group, received intracavernous injection of saline, group 2,the VEGF group, received intracavernous injection of 4 ug of VEGF, andgroup 3, the AAV-BDNF group received 15 ug of BDNF. After six months ofcholesterol diet and 4 months of treatment, all rats were subjected tocavernous nerve electrostimulation, cavemosometry with intracavernouspapaverine injection, and infusion cavernosometry with heparinizedsaline to measure erectile function.

Results

[0382] Serum cholesterol levels were significantly higher in animals fedthe high cholesterol diet. Systemic arterial pressure was notsignificantly different among the different groups. Duringelectrostimulation of the cavernous nerve, peak sustained intracavernouspressure was significantly lower in the cholesterol only group (50+/−23cm. H₂O) compared to the control and the VEFG and AAV-BDNF groups.During the pharmacologic erection phase of the cavernosometry, the VEGFand BDNF treated groups had significantly higher sustainedintracavernous pressures in comparison to the high cholesterol controls.No difference was noted with respect to the infusion cavernosometryassessing venous leak when comparing the controls and the treatedgroups.

Conclusion

[0383] Rats developed erectile dysfunction after being fed a highcholesterol diet (2.5% with lard) for 6 months. VEGF and AAV-BDNF seemto reverse the erectile dysfunction caused by high cholesterol diet.

EXAMPLE 6

[0384] The Effect of Intracavernous Vascular Endothelial Growth Factor(VEGF) on a Rodent Model of Neurogenic Erectile Dysfunction

[0385] Objective

[0386] To test the hypothesis that intracavernous injection of VEGF canfacilitate regeneration of the cavernous nerve and restore erectilefunction after cavernous nerve injury in rats

[0387] Materials and Methods

[0388] Seventeen 3 months old Sprague Dowry rats underwent bilateralfreezing of the cavernous nerves with a thermocouple immersed in liquidnitrogen. Through an abdominal incision both cavernous nerves wereisolated lateral to the prostate and frozen with thermocouple for oneminute twice (temperature cycle −130° C. to −3° C.). Minutes later,intracavernous injection of saline (n=7) or VEGF, 4 ug, (n=9) was given.Three months later, all rats underwent re-exploration andelectrostimulation of the cavernous nerves to assess erectile function.Eight additional rats underwent exploration only (sham group)

Results

[0389] The maximal intracavernous pressure in the rats underwent shamoperation was 90±8.15 cm H2O. The maximal intracavernous pressure ofboth the saline treated group (39.29±5.02 cm H₂O) and the VEGF treatedgroup (72.78±10.87 cm H₂O) were lower than the sham group. Nevertheless,the intracavernous pressure in the VEGF treated group was significantlyhigher than the saline treated group (p=0.0389).

Conclusion

[0390] Although VEGF is known as an angiogenetic factor, previousreports have suggested that it may also be neuroprotective andneurotrophic. Our study shows that intracavernous injection of VEGFsignificantly facilitated recovery of erectile function after bilateralcavernous nerve injury. If further studies confirmed that VEGF enhancesboth angiogenesis and neural regeneration, intracavernous VEGF therapymay be the treatment of choice in helping patient recover potency afterradical prostatectomy or cryoablation of the prostate.

EXAMPLE 7 Synergistic Neurotrophic Effects of Vascular EndothelialGrowth Factor and Brain-Derived Neurotrophic Factor

[0391] Neurogenic impotence due to cavernous nerve injury occursfrequently in patients having undergone radical surgeries for prostate,bladder and rectal cancer. To investigate the feasibility of an in vitroassay system for nerve regeneration, we devised a model in which themajor pelvic ganglia (MPG), from which the cavernous nerves areoriginated, were isolated and grown in culture.

[0392] Materials and Methods

[0393] Each freshly isolated MPG was cut into 8 pieces of approximatelyequal size and each piece was then attached to a Matrigel-coatedcoverslip. The MPG pieces were cultured in serum-free mediumsupplemented with phosphate-buffered saline (PBS, control), 50 ng/ml ofvascular endothelial growth factor (VEGF), 20 ng/ml of brain-derivedneurotrophic factor (BDNF), or VEGF+BDNF. After 2 days of incubation,the ganglial tissues with their outgrowing nerve fibers were stained forthe expression of NADPH diaphorase and acetylcholinesterase (AchE).

[0394] Results

[0395] The average lengths of the outgrowing nerve fibers weredetermined to be 16±7 nm, 89±21 nm, 75±11 nm and 124±23 nm, for tissuestreated with PBS, VEGF, BDNF, and VEGF+BDNF, respectively (Table 10).The levels of NADPH diaphorase expression, calculated as integrateddensity value (IDV), were 2,010±82, 10,126±187, 9,376±111 and 13,616±210for tissues treated with PBS, VEGF, BDNF and VEGF+BDNF, respectively.The levels of AchE expression were 29±2, 8,470±199, 1,102±211 and11,006±198 for tissues treated with PBS, VEGF, BDNF and VEGF+BDNF,respectively. TABLE 10 Growth Average fiber NADPH staining AchE stainingfactor length, in nin intensity* intensity* VEGF  89 ± 21 10,126 ± 187 8,470 ± 199 BDNF  75 ± 11  9,376 ± 111  1,102 ± 211 VEGF 124 ± 2313,616 ± 210 11,006 ± 198 + BDNF Control  16 ± 7  2,010 ± 82   29 ± 2(PBS)

Conclusions

[0396] These results indicate that both VEGF and BDNF had neurotrophiceffects and were synergistic in combination. In addition, it appearsthat BDNF preferentially promoted the growth of NOS-containing nerves(positive for NADPH diaphorase), as opposed to cholinergic nerves(AchE-positive).

EXAMPLE 8 Synergistic Neurotrophic Effects of Vascular EndothelialGrowth Factor and Neurotrophins

[0397] We have been using several experimental systems to test theefficacy of combination growth factor therapies for impotence. Onesystem employs cultured rat major pelvic ganglia (MPG) that are known toinnervate all urogenital organs, including the penis. We discovered thatthe cultured MPG are able to sprout new nerve fibers (nerveregeneration) from the axonal ends during the first 3 days of culture,especially when treated with VEGF, BDNF, NT-3, or NT-4. The averagelengths (in riM) of the growing fibers 48 hours after culturing withvarious treatments are listed in Table 11. TABLE 11 GROWTH FACTORS FIBERLENGTH VEGF 89 BDNF 75 NT-3 112 NT-4 81 VEGF + BDNF 124 VEGF + NT-3 132VEGF ± NT-4 101 BDNF ± NT-3 88 BDNF + NT-4 81 Control (treated withsaline) 16

[0398] Compared with an average length of 16 nm in control MPG, it isclear that VEGF, BDNF, NT-3, and NT-4 all have neurotrophic effects.More importantly, when VEGF is combined with each of the threeneurotrophins, there was a synergistic effect.

EXAMPLE 9 VEGF-Enhanced Neurotrophin Therapy (VENT)

[0399] This example shows the testing of the hypothesis thatintracavernous injection of vascular endothelial growth factor (VEGF)enhances the effect of brain derived neurotrophic factor (BDNF) and thusfacilitate neural and erectile function recovery after cavernous nerveinjury.

[0400] Materials and Methods

[0401] Three months old Sprague-Dawley rats (N=30) were used: 6underwent a sham operation; 24 underwent bilateral cavernous nervefreezing and then randomly divided into four groups of 6 each. The fourgroups of rats received intracavernous injection of one of thefollowing: 1) phosphate buffered saline (PBS), 2) VEGF protein (4 μg),3) adeno-associated virus (AAV)-BDNF, and 4). VEGF+AAV-BDNF. Erectilefunction was assessed by cavernous nerve electrostimulation 3 monthslater and samples of penile tissue were obtained for electron microscopyand immunohistochemical studies.

[0402] Results

[0403] The maximal intracavernous pressure (MICP) of the rats (mean±SEMcm H2O) in the sham, PBS, VEGF, BDNF and VEGF +BDNF groups were100.67±6.55, 29.25±3.90, 37.33±3.29, 69.75±8.54, 87.17±6.00respectively. The differences were statistically significant between thesham and all other groups but not between sham and the VEGF +BDNF group.The blood vessel +capillary count was decreased in the PBS group (3±1.6vs. 6±1.3 in sham group) but was significant higher in the VDGF+BDNFgroup (9.8±2.3, p=0.01 vs. PBS group). Image analysis of nNOS stainingrevealed significantly less NNOS positive nerve fibers in the dorsalnerves in the PBS groups as compared to the sham group. There was anincreased number of positive nerve fibers in VEGF, BDNF and VEGF+BDNFgroups as compared to PBS group. Electron microscopy revealed that theVEGF+BDNF group had the best overall morphology of both myelinated andnonmyelinated nerve fibers in the penis.

Conclusions

[0404] Intracavemous injection of VEGF and AAV-BDNF improved theregeneration of nerve fibers in the penis and the recovery of erectilefunction. The best results were obtained with intracavernous injectionof both VEGF and AAV-BDNF. The increased endothelial permeability ofVEGF may facilitate the transport of itself and AAV-BDNF to the erectiletissue and thus enhance the neurotrophic and angiogenic effect.

EXAMPLE 10 A New Model for Angiogenesis Studies

[0405] Penile dorsal arteries were isolated from rats and cut into 1-mmsegments under a 10× microscope. Each segment was attached to aMatrigel-coated coverslip, which was then put in a 60-mm culture dish.Serum-free medium with or without VEGF was added and the arterialculture was incubated for 3 days. The results demonstrate the budding ofnew blood vessels that were significantly longer in VEGF-treated than inuntreated culture. The budding vessels were stained positive with RECAantibody, indicating the endothelial identity. To our best knowledge,this is the first demonstration of angiogenesis from blood vesselexplants. The ease of this experimental procedure in comparison withexisting methods should greatly facilitate future angiogenesis studies.

EXAMPLE 11 Artery-Muscle Co-Culture Systems

[0406] We extended our above angiogenesis model to include muscles in aco-culture system. The source of muscle was corpus cavernosum, urethrasphincter, or bladder. In each case, the outgrowing blood vessels showedattraction to and seemingly entering the muscle. It also appears thatVEGF encourage the growth of endothelial cells and VEGF+PDGF stimulatedthe growth of both endothelial and smooth muscle cells. In addition, thecombination of VEGF and PDGF encouraged more extensive contacts betweenthe outgrowing vessels with the muscle. More studies with either singleor a combination of other growth factors and the molecular mechanism ofvessel growth are currently underway. Once the best growth factor orcombinations are identified, we will perform in vivo studies in ratswith severe vasculogenic ED to see if the in vitro assay helps identifythe best angiogenins.

[0407] The examples set forth above are provided to give those ofordinary skill in the art with a complete disclosure and description ofhow to make and use the preferred embodiments of the compositions, andare not intended to limit the scope of what the inventors regard astheir invention. Modifications of the above-described modes for carryingout the invention that are obvious to persons of skill in the art areintended to be within the scope of the following claims. Allpublications, patents, and patent applications cited in thisspecification are incorporated herein by reference as if each suchpublication, patent or patent application were specifically andindividually indicated to be incorporated herein by reference.

1 16 1 21 DNA Artificial Sequence Primer 1 ccctacaggt cgaccaggtg a 21 221 DNA Artificial Sequence Primer 2 ctatacaaca tggatccact a 21 3 20 DNAArtificial Sequence Primer Actin-s 3 tctacaatga gctgcgtgtg 20 4 20 DNAArtificial Sequence Primer Actin-a 4 ccctttagca cgcactgtaa 20 5 19 DNAArtificial Sequence Primer VEGFR-1s 5 atgctggatt gctggcaca 19 6 20 DNAArtificial Sequence Primer VEGFR-1a 6 ttacggtgga ggtacaaact 20 7 20 DNAArtificial Sequence Primer VEGFR-2s 7 gcctttggcc aagtgattga 20 8 20 DNAArtificial Sequence Primer VEGFR-2a 8 ttcctgaagg actggaacct 20 9 990 DNAArtificial Sequence Synthetic construct 9 cagtgtgctg gcggcccggcgcgagccggc ccggccccgg tcgggcctcc gaaaccatga 60 actttctgct gtcttgggtgcattggagcc tcgccttgct gctctacctc caccatgcca 120 agtggtccca ggctgcacccatggcagaag gaggagggca gaatcatcac gaagtggtga 180 agttcatgga tgtctatcagcgcagctact gccatccaat cgagaccctg gtggacatct 240 tccaggagta ccctgatgagatcgagtaca tcttcaagcc atcctgtgtg cccctgatgc 300 gatgcggggg ctgctgcaatgacgagggcc tggagtgtgt gcccactgag gagtccaaca 360 tcaccatgca gattatgcggatcaaacctc accaaggcca gcacatagga gagatgagct 420 tcctacagca caacaaatgtgaatgcagac caaagaaaga tagagcaaga caagaaaatc 480 cctgtgggcc ttgctcagagcggagaaagc atttgtttgt acaagatccg cagacgtgta 540 aatgttcctg caaaaacacagactcgcgtt gcaaggcgag gcagcttgag ttaaacgaac 600 gtacttgcag atgtgacaagccgaggcggt gagccgggca ggaggaagga gcctccctca 660 gggtttcggg aaccagatctctcaccagga aagactgata cagaacgatc gatacagaaa 720 ccacgctgcc gccaccacaccatcaccatc gacagaacag tccttaatcc agaaacctga 780 aatgaaggaa gaggagactctgcgcagagc actttgggtc cggagggcga gactccggcg 840 gaagcattcc cgggcgggtgacccagcacg gtccctcttg gaattggatt cgccatttta 900 tttttcttgc tgctaaatcaccgagcccgg aagattagag agttttattt ctgggattcc 960 tgtagacaca ccgcggccgccagcacactg 990 10 1574 DNA Artificial Sequence Synthetic construct 10gttccccaac tgctgtttta ttgtgctatt catgcctaga catcacatag ctagaaaggc 60ccatcagacc cctcaggcca ctgctgttcc tgtcacacat tcctgcaaag gaccatgttg 120ctaacttgaa aaaaattact attaattaca cttgcagttg ttgcttagta acatttatga 180ttttgtgttt ctcgtgacag catgagcaga gatcattaaa aattaaactt acaaagctgc 240taaagtggga agaaggagaa cttgaagcca caatttttgc acttgcttag aagccatcta 300atctcaggtt atatgctaga tcttgggggc aaacactgca tgtctctggt ttatattaaa 360ccacatacag cacactactg acactgattt gtgtctggtg cagctggagt ttatcaccaa 420gacataaaaa aaccttgacc ctgcagaatg gcctggaatt acaatcagat gggccacatg 480gcatcccggt gaaagaaagc cctaaccagt tttctgtctt gtttctgctt tctccctaca 540gttccaccag gtgagaagag tgatgaccat ccttttcctt actatggtta tttcatactt 600tggttgcatg aaggctgccc ccatgaaaga agcaaacatc cgaggacaag gtggcttggc 660ctacccaggt gtgcggaccc atgggactct ggagagcgtg aatgggccca aggcaggttc 720aagaggcttg acatcattgg ctgacacttt cgaacacgtg atagaagagc tgttggatga 780ggaccagaaa gttcggccca atgaagaaaa caataaggac gcagacttgt acacgtccag 840ggtgatgctc agtagtcaag tgcctttgga gcctcctctt ctctttctgc tggaggaata 900caaaaattac ctagatgctg caaacatgtc catgagggtc cggcgccact ctgaccctgc 960ccgccgaggg gagctgagcg tgtgtgacag tattagtgag tgggtaacgg cggcagacaa 1020aaagactgca gtggacatgt cgggcgggac ggtcacagtc cttgaaaagg tccctgtatc 1080aaaaggccaa ctgaagcaat acttctacga gaccaagtgc aatcccatgg gttacacaaa 1140agaaggctgc aggggcatag acaaaaggca ttggaactcc cagtgccgaa ctacccagtc 1200gtacgtgcgg gcccttacca tggatagcaa aaagagaatt ggctggcgat tcataaggat 1260agacacttct tgtgtatgta cattgaccat taaaagggga agatagtgga tttatgttgt 1320atagattaga ttatattgag acaaaaatta tctatttgta tatatacata acagggtaaa 1380ttattcagtt aagaaaaaaa taattttatg aactgcatgt ataaatgaag tttatacagt 1440acagtggttc tacaatctat ttattggaca tgtccatgac cagaagggaa acagtcattt 1500gcgcacaact taaaaagtct gcattacatt ccttgataat gttgtggttt gttgccgttg 1560ccaagaactg aaaa 1574 11 444 DNA Artificial Sequence Synthetic construct11 atgccagcat tgcccgagga tggcggcagc ggcgccttcc cgcccggcca cttcaaggac 60cccaagcggc tgtactgcaa aaacgggggc ttcttcctgc gcatccaccc cgacggccga 120gttgacgggg tccgggagaa gagcgaccct cacatcaagc tacaacttca agcagaagag 180agaggagttg tgtctatcaa aggagtgtgt gctaaccgtt acctggctat gaaggaagat 240ggaagattac tggcttctaa atgtgttacg gatgagtgtt tcttttttga acgattggaa 300tctaataact acaatactta ccggtcaagg aaatacacca gttggtatgt ggcactgaaa 360cgaactgggc agtataaact tggatccaaa acaggacctg ggcagaaagc tatacttttt 420cttccaatgt ctgctaagag ctga 444 12 1020 DNA Artificial Sequence Syntheticconstruct 12 taacacagac tcagctgcca gagcctgctc ttaacacctg tgtttccttttcagatctta 60 caggtgaaca aggtgatgtc catcttgttt tatgtgatat ttctcgcttatctccgtggc 120 atccaaggta acaacatgga tcaaaggagt ttgccagaag actcgctcaattccctcatt 180 attaagctga tccaggcaga tattttgaaa aacaagctct ccaagcagatggtggacgtt 240 aaggaaaatt accagagcac cctgcccaaa gctgaggctc cccgagagccggagcgggga 300 gggcccgcca agtcagcatt ccagccggtg attgcaatgg acaccgaactgctgcgacaa 360 cagagacgct acaactcacc gcgggtcctg ctgagcgaca gcacccccttggagcccccg 420 cccttgtatc tcatggagga ttacgtgggc agccccgtgg tggcgaacagaacatcacgg 480 cggaaacggt acgcggagca taagagtcac cgaggggagt actcggtatgtgacagtgag 540 agtctgtggg tgaccgacaa gtcatcggcc atcgacattc ggggacaccaggtcacggtg 600 ctgggggaga tcaaaacggg caactctccc gtcaaacaat atttttatgaaacgcgatgt 660 aaggaagcca ggccggtcaa aaacggttgc aggggtattg atgataaacactggaactct 720 cagtgcaaaa catcccaaac ctacgtccga gcactgactt cagagaacaataaactcgtg 780 ggctggcggt ggatacggat agacacgtcc tgtgtgtgtg ccttgtcgagaaaaatcgga 840 agaacatgaa ttggcatctc tccccatata taaattatta ctttaaattatatgatatgc 900 atgtagcata taaatgttta tattgttttt atatattata agttgacctttatttattaa 960 acttcagcaa ccctacagta tataagcttt tttctcaata aaatcagtgtgcttgccttc 1020 13 1404 DNA Artificial Sequence Synthetic construct 13cttgtcaccc aggtggcagg ggagtggtgc actctctgct cactgcaacc tcggcctcct 60gggttcgagt gattctccta cctcagccta ctgagtagct gggattacag gcgtgcagca 120ctatgcccgg ttaattttgg tatttttggt agagatgagg tttcaccatg ttgaccagct 180gctctggaac tcctgacctc aagtcatcca cctgcctcag cctcccagag tgctgggatt 240agaggtgtgg ggcacagtgc ctggcctgta gtagttgaat atttattatt aatctacaag 300ttgcgcatta cgcaagccct agatataggg tcccccaaac ttctagaaca agggcttccc 360cacaatcctg gcaggcaagc ctcccctggg gttcccaact tctttcccca ctgaagtttt 420tacccccttc tctaatccca gcctccctct ttctgtctcc aggtgctccg agagatgctc 480cctctcccct catgctccct ccccatcctc ctccttttcc tcctccccag tgtgccaatt 540gagtcccaac ccccaccctc aacattgccc ccttttctgg cccctgagtg ggaccttctc 600tccccccgag tagtcctgtc taggggtgcc cctgctgggc cccctctgct cttcctgctg 660gaggctgggg cctttcggga gtcagcaggt gccccggcca accgcagccg gcgtggggtg 720agcgaaactg caccagcgag tcgtcggggt gagctggctg tgtgcgatgc agtcagtggc 780tgggtgacag accgccggac cgctgtggac ttgcgtgggc gcgaggtgga ggtgttgggc 840gaggtgcctg cagctggcgg cagtcccctc cgccagtact tctttgaaac ccgctgcaag 900gctgataacg ctgaggaagg tggcccgggg gcaggtggag ggggctgccg gggagtggac 960aggaggcact gggtatctga gtgcaaggcc aagcagtcct atgtgcgggc attgaccgct 1020gatgcccagg gccgtgtggg ctggcgatgg attcgaattg acactgcctg cgtctgcaca 1080ctcctcagcc ggactggccg ggcctgagac ccatgcccag gaaaataaca gagctggatg 1140ctgagagacc tcagggatgg cccagctgat ctaaggaccc cagtttggga actcatcaaa 1200taatcacaaa atcacaattc tctgattttg agctcaatct ctgcaggatg ggtgaaacca 1260catggggttt tggaggttga ataggagttc tcctggagca acttgagggt aataatgatg 1320atgatataat aataatagcc actatttact gagtgtttac tgtttcttat ccctaataca 1380taactcctca gatcaactct catg 1404 14 2137 DNA Artificial SequenceSynthetic construct 14 ccctgcctgc ctccctgcgc acccgcagcc tcccccgctgcctccctagg gctcccctcc 60 ggccgccagc gcccattttt cattccctag atagagatactttgcgcgca cacacataca 120 tacgcgcgca aaaaggaaaa aaaaaaaaaa aagcccaccctccagcctcg ctgcaaagag 180 aaaaccggag cagccgcagc tcgcagctcg cagcccgcagcccgcagagg acgcccagag 240 cggcgagcgg gcgggcagac ggaccgacgg actcgcgccgcgtccacctg tcggccgggc 300 ccagccgagc gcgcagcggg cacgccgcgc gcgcggagcagccgtgcccg ccgcccgggc 360 ccgccgccag ggcgcacacg ctcccgcccc cctacccggcccgggcggga gtttgcacct 420 ctccctgccc gggtgctcga gctgccgttg caaagccaactttggaaaaa gttttttggg 480 ggagacttgg gccttgaggt gcccagctcc gcgctttccgattttggggg cctttccaga 540 aaatgttgca aaaaagctaa gccggcgggc agaggaaaacgcctgtagcc ggcgagtgaa 600 gacgaaccat cgactgccgt gttccttttc ctcttggaggttggagtccc ctgggcgccc 660 ccacacggct agacgcctcg gctggttcgc gacgcagccccccggccgtg gatgctgcac 720 tcgggctcgg gatccgccca ggtagcggcc tcggacccaggtcctgcgcc caggtcctcc 780 cctgcccccc agcgacggag ccggggccgg gggcggcggcgccgggggca tgcgggtgag 840 ccgcggctgc agaggcctga gcgcctgatc gccgcggacccgagccgagc ccacccccct 900 ccccagcccc ccaccctggc cgcgggggcg gcgcgctcgatctacgcgtt cggggccccg 960 cggggccggg cccggagtcg gcatgaatcg ctgctgggcgctcttcctgt ctctctgctg 1020 ctacctgcgt ctggtcagcg ccgaggggga ccccattcccgaggagcttt atgagatgct 1080 gagtgaccac tcgatccgct cctttgatga tctccaacgcctgctgcacg gagaccccgg 1140 agaggaagat ggggccgagt tggacctgaa catgacccgctcccactctg gaggcgagct 1200 ggagagcttg gctcgtggaa gaaggagcct gggttccctgaccattgctg agccggccat 1260 gatcgccgag tgcaagacgc gcaccgaggt gttcgagatctcccggcgcc tcatagaccg 1320 caccaacgcc aacttcctgg tgtggccgcc ctgtgtggaggtgcagcgct gctccggctg 1380 ctgcaacaac cgcaacgtgc agtgccgccc cacccaggtgcagctgcgac ctgtccaggt 1440 gagaaagatc gagattgtgc ggaagaagcc aatctttaagaaggccacgg tgacgctgga 1500 agaccacctg gcatgcaagt gtgagacagt ggcagctgcacggcctgtga cccgaagccc 1560 ggggggttcc caggagcagc gagccaaaac gccccaaactcgggtgacca ttcggacggt 1620 gcgagtccgc cggcccccca agggcaagca ccggaaattcaagcacacgc atgacaagac 1680 ggcactgaag gagacccttg gagcctaggg gcatcggcaggagagtgtgt gggcagggtt 1740 atttaatatg gtatttgctg tattgccccc atggggccttggagtagata atattgtttc 1800 cctcgtccgt ctgtctcgat gcctgattcg gacggccaatggtgcctccc ccacccctcc 1860 acgtgtccgt ccacccttcc atcagcgggt ctcctcccagcggcctccgg ctcttgccca 1920 gcagctcaag aagaaaaaga aggactgaac tccatcgccatcttcttccc ttaactccaa 1980 gaacttggga taagagtgtg agagagactg atggggtcgctctttggggg aaacgggttc 2040 cttcccctgc acctggcctg ggccacacct gagcgctgtggactgtcctg aggagccctg 2100 aggacctctc agcatagcct gcctgatccc tgaaccc 213715 1308 DNA Artificial Sequence Synthetic construct 15 tccgcaaatatgcagaatta ccggccgggt cgctcctgaa gccagcgcgg ggaggcagcg 60 cggcggcggccagcaccggg aacgcaccga ggaagaagcc cagcccccgc cctccgcccc 120 ttccgtccccacccccatcc cggcggccca ggaggctccc cgcgctggcg cgcactccct 180 gtttctcctcctcctggctg gcgctgcctg cctctccgca ctcactgctc gccgggcgcc 240 gtccgccagctccgtgctcc ccgcgccacc ctcctccggg ccgcgctccc taagggatgg 300 tactgattttcgccgccaca ggagaccggc tggagcgccg ccccgcggcc tcgcctctcc 360 tccgagcagccagcgcctcg ggacgcgatg aggaccttgg cttgcctgct gctcctcggc 420 tgcggatacctcgcccatgt tctggccgag gaagccgaga tcccccgcga ggtgatcgag 480 aggctggcccgcagtcagat ccacagcatc cgggacctcc agcgactcct ggagatagac 540 tccgtagggagtgaggattc tttggacacc agcctgagag ctcacggggt ccatgccact 600 aagcatgtgcccgagaagcg gcccctgccc attcggagga agagaagcat cgaggaagct 660 gtccccgctgtctgcaagac caggacggtc atttacgaga ttcctcggag tcaggtcgac 720 cccacgtccgccaacttcct gatctggccc ccgtgcgtgg aggtgaaacg ctgcaccggc 780 tgctgcaacacgagcagtgt caagtgccag ccctcccgcg tccaccaccg cagcgtcaag 840 gtggccaaggtggaatacgt caggaagaag ccaaaattaa aagaagtcca ggtgaggtta 900 gaggagcatttggagtgcgc ctgcgcgacc acaagcctga atccggatta tcgggaagag 960 gacacgggaaggcctaggga gtcaggtaaa aaacggaaaa gaaaaaggtt aaaacccacc 1020 taaagcagccaaccagatgt gaggtgagga tgagccgcag ccctttcctg ggacatggat 1080 gtacatggcgtgttacattc ctgaacctac tatgtacggt gctttattgc cagtgtgcgg 1140 tctttgttctcctccgtgaa aaactgtgtc cgagaacact cgggagaaca aagagacagt 1200 gcacatttgtttaatgtgac atcaaagcaa gtattgtagc actcggtgaa gcagtaagaa 1260 gcttccttgtcaaaaagaga gagagagaaa agaaaaaaaa aggaattc 1308 16 2149 DNA ArtificialSequence Synthetic construct 16 cagctgactc aggcaggctc catgctgaacggtcacacag agaggaaaca ataaatctca 60 gctactatgc aataaatatc tcaagttttaacgaagaaaa acatcattgc agtgaaataa 120 aaaattttaa aattttagaa caaagctaacaaatggctag ttttctatga ttcttcttca 180 aacgctttct ttgaggggga aagagtcaaacaaacaagca gttttacctg aaataaagaa 240 ctagttttag aggtcagaag aaaggagcaagttttgcgag aggcacggaa ggagtgtgct 300 ggcagtacaa tgacagtttt cctttcctttgctttcctcg ctgccattct gactcacata 360 gggtgcagca atcagcgccg aagtccagaaaacagtggga gaagatataa ccggattcaa 420 catgggcaat gtgcctacac tttcattcttccagaacacg atggcaactg tcgtgagagt 480 acgacagacc agtacaacac aaacgctctgcagagagatg ctccacacgt ggaaccggat 540 ttctcttccc agaaacttca acatctggaacatgtgatgg aaaattatac tcagtggctg 600 caaaaacttg agaattacat tgtggaaaacatgaagtcgg agatggccca gatacagcag 660 aatgcagttc agaaccacac ggctaccatgctggagatag gaaccagcct cctctctcag 720 actgcagagc agaccagaaa gctgacagatgttgagaccc aggtactaaa tcaaacttct 780 cgacttgaga tacagctgct ggagaattcattatccacct acaagctaga gaagcaactt 840 cttcaacaga caaatgaaat cttgaagatccatgaaaaaa acagtttatt agaacataaa 900 atcttagaaa tggaaggaaa acacaaggaagagttggaca ccttaaagga agagaaagag 960 aaccttcaag gcttggttac tcgtcaaacatatataatcc aggagctgga aaagcaatta 1020 aacagagcta ccaccaacaa cagtgtccttcagaagcagc aactggagct gatggacaca 1080 gtccacaacc ttgtcaatct ttgcactaaagaaggtgttt tactaaaggg aggaaaaaga 1140 gaggaagaga aaccatttag agactgtgcagatgtatatc aagctggttt taataaaagt 1200 ggaatctaca ctatttatat taataatatgccagaaccca aaaaggtgtt ttgcaatatg 1260 gatgtcaatg ggggaggttg gactgtaatacaacatcgtg aagatggaag tctagatttc 1320 caaagaggct ggaaggaata taaaatgggttttggaaatc cctccggtga atattggctg 1380 gggaatgagt ttatttttgc cattaccagtcagaggcagt acatgctaag aattgagtta 1440 atggactggg aagggaaccg agcctattcacagtatgaca gattccacat aggaaatgaa 1500 aagcaaaact ataggttgta tttaaaaggtcacactggga cagcaggaaa acagagcagc 1560 ctgatcttac acggtgctga tttcagcactaaagatgctg ataatgacaa ctgtatgtgc 1620 aaatgtgccc tcatgttaac aggaggatggtggtttgatg cttgtggccc ctccaatcta 1680 aatggaatgt tctatactgc gggacaaaaccatggaaaac tgaatgggat aaagtggcac 1740 tacttcaaag ggcccagtta ctccttacgttccacaacta tgatgattcg acctttagat 1800 ttttgaaagc gcaatgtcag aagcgattatgaaagcaaca aagaaatccg gagaagctgc 1860 caggtgagaa actgtttgaa aacttcagaagcaaacaata ttgtctccct tccagcaata 1920 agtggtagtt atgtgaagtc accaaggttcttgaccgtga atctggagcc gtttgagttc 1980 acaagagtct ctacttgggg tgacagtgctcacgtggctc gactatagaa aactccactg 2040 actgtcgggc tttaaaaagg gaagaaactgctgagcttgc tgtgcttcaa actactactg 2100 gaccttattt tggaactatg gtagccagatgataaatatg gttaatttc 2149

1. A method for preventing or treating male erectile dysfunction or female sexual arousal disorder, which method comprises administering to a mammal to whom such prevention or treatment is needed or desirable, an effective amount of a factor, wherein the factor is selected from the group consisting of vascular endothelial growth factor (VEGF), brain-derived growth factor (BDNF), basic fibroblast growth factor (bFGF), neurotrophin-3 (NT-3), neurotrophin-4 (NT-4), platelet-derived growth factor (PDGF), angiopoietin-1 (Ang-1), an agent that enhances production and/or male erection or female sexual arousal stimulating function of the factor, and a combination thereof, thereby preventing or treating the male erectile dysfunction or the female sexual arousal disorder in the mammal.
 2. The method of claim 1, wherein the factor is a full length protein or a functional derivative or fragment thereof, or a nucleic acid encoding the factor or functional derivative or fragment thereof, or an agent that enhances production and/or male erection or female sexual arousal stimulating function of the factor.
 3. The method of claim 1, wherein the mammal is a human and the factor, or a functional derivative or fragment thereof, or the nucleic acid encoding the factor, or a functional derivative or fragment thereof, is of human origin.
 4. The method of claim 1, wherein the factor protein or nucleic acid, or a functional derivative or fragment thereof, is administered by intracavemous injection, subcutaneous injection, intravenous injection, intramuscular injection, intradermal injection, or topical administration.
 5. The method of claim 1, wherein the factor nucleic acid, or a functional derivative or fragment thereof, is administered via a gene therapy vector.
 6. The method of claim 5, wherein the gene therapy vector is selected from the group consisting of an adenovirus associated vector, a retroviral vector, an adenovirus vector, and a lentivirus vector.
 7. The method of claim 6, wherein the gene therapy vector is an adenovirus associated vector.
 8. The method of claim 1, wherein the factor protein, or a functional derivative or fragment thereof, is administered via a liposome.
 9. The method of claim 1, wherein the factor nucleic acid, or a functional derivative or fragment thereof, is administered via a liposome.
 10. The method of claim 1, wherein the male erectile dysfunction is erectile dysfunction induced by or secondary to nerve dysfunction, arterial insufficiency, venous leakage, severe vascular insufficiency, hormonal insufficiency, drug use, surgery, chemotherapy, or radiation.
 11. The method of claim 1, wherein the female sexual arousal disorder is sexual dysfunction induced by or secondary to nerve dysfunction, arterial insufficiency, severe vascular, insufficiency, hormonal insufficiency, drug use, surgery, chemotherapy, or radiation.
 12. The method of claim 1, wherein the factor protein or a functional derivative or fragment thereof, or a nucleic acid encoding the factor or functional derivative or fragment thereof, or an agent that enhances production and/or female sexual arousal stimulating function of the factor, is administered in an amount sufficient to improve blood flow and regenerate nerve and smooth muscle in the clitoris and vaginal wall.
 13. The method of claim 1, wherein the factor protein or a functional derivative or fragment thereof, or a nucleic acid encoding the factor or functional derivative or fragment thereof, or an agent that enhances production and/or female sexual arousal stimulating function of said factor, is administered in a cream or via injection to the clitoris and vaginal wall of the patient.
 14. The method of claim 1, wherein the factor protein or a functional derivative or fragment thereof, or a nucleic acid encoding the factor or functional derivative or fragment thereof, or an agent that enhances production and/or the male erection or female sexual arousal stimulating function of the factor, is administered by intracavemous injection.
 15. The method of claim 1, wherein the male erectile dysfunction or female sexual arousal disorder is induced by or secondary to nerve injury.
 16. The method of claim 15, wherein the combination of factors are a) VEGF and NT-3, b) VEGF and NT-4, or c) VEGF and BDNF.
 17. The method of claim 1, wherein the male erectile dysfunction or female sexual arousal disorder is induced by or secondary to severe vascular insufficiency.
 18. The method of claim 17, wherein the combination of factors are: a) VEGF and PDGF, b) VEGF and bFGF, or c) VEGF and Ang-1.
 19. The method of claim 1, wherein the male erectile dysfunction or female sexual arousal disorder is induced by or secondary to mild vascular disease.
 20. The method of claim 19, wherein the factor is VEGF.
 21. A combination for preventing or treating male erectile dysfunction or female sexual arousal disorder, which combination comprises: a) an effective amount of an agent that stimulates male erectile or female sexual function; and b) an effective amount of a factor, wherein the factor is a full length protein or a functional derivative or fragment thereof, or a nucleic acid encoding said factor or functional derivative or fragment thereof, or an agent that enhances production and/or male erection or female sexual arousal stimulating function of said factor and said factor is selected from the group consisting of vascular endothelial growth factor (VEGF), brain-derived growth factor (BDNF), basic fibroblast growth factor (bFGF), neurotrophin-3 (NT-3), neurotrophin-4 (NT-4), platelet-derived growth factor (PDGF), and angiopoietin-1 (Ang-1).
 22. The combination of claim 21, wherein the combination is in the form of a pharmaceutical composition.
 23. The combination of claim 22, which further comprises a pharmaceutically acceptable carrier or excipient.
 24. A combination for preventing or treating male erectile dysfunction or female sexual arousal disorder induced by or secondary to nerve injury, which combination comprises: a) an effective amount of VEGF, wherein the VEGF is a full length protein or a functional derivative or fragment thereof, or a nucleic acid encoding the VEGF or functional derivative or fragment thereof; and b) an effective amount of a factor selected from the group consisting of NT-3, NT-4, and BDNF, wherein the factor is a full length protein or a functional derivative or fragment thereof, or a nucleic acid encoding the factor or functional derivative or fragment thereof.
 25. The combination of claim 24, wherein the combination is in the form of a pharmaceutical composition.
 26. The combination of claim 25, which further comprises a pharmaceutically acceptable carrier or excipient.
 27. A combination for preventing or treating male erectile dysfunction or female sexual arousal disorder induced by or secondary to severe vascular insufficiency, which combination comprises: a) an effective amount of VEGF, wherein the VEGF is a full length protein or a functional derivative or fragment thereof, or a nucleic acid encoding the VEGF or functional derivative or fragment thereof; and b) an effective amount of a factor selected from the group consisting of PDGF, bFGF, and Ang-1, wherein the factor is a full length protein or a functional derivative or fragment thereof, or a nucleic acid encoding the factor or functional derivative or fragment thereof.
 28. The combination of claim 27, wherein the combination is in the form of a pharmaceutical composition.
 29. The combination of claim 28, which further comprises a pharmaceutically acceptable carrier or excipient.
 30. A method for preventing or treating male erectile dysfunction or female sexual arousal disorder, which method comprises administering an effective amount of the combination of claim 21 to a mammal in need thereof, thereby preventing or treating the male erectile dysfunction or the female sexual arousal disorder in the mammal.
 31. A method for preventing or treating male erectile dysfunction or female sexual arousal disorder, which method comprises administering an effective amount of the combination of claim 24 to a mammal in need thereof, thereby preventing or treating the male erectile dysfunction or the female sexual arousal disorder in the mammal.
 32. A method for preventing or treating male erectile dysfunction or female sexual arousal disorder, which method comprises administering an effective amount of the combination of claim 27 to a mammal in need thereof, thereby preventing or treating the male erectile dysfunction or the female sexual arousal disorder in the mammal.
 33. A kit, which kit comprises the combination of claim 21 and an instruction for using the combination in treating or preventing male erectile dysfunction or female sexual arousal disorder.
 34. A kit, which kit comprises the combination of claim 24 and an instruction for using the combination in treating or preventing male erectile dysfunction or female sexual arousal disorder.
 35. A kit, which kit comprises the combination of claim 27 and an instruction for using the combination in treating or preventing male erectile dysfunction or female sexual arousal disorder.
 36. A method of promoting sprouting of new nerve fibers from blood vessel explants, which method comprises the steps of: a) isolating a blood vessel; b) attaching said vessel to a media-coated covership; and c) incubating with a growth-stimulating compound.
 37. The method of claim 36, wherein the compound is VEGF.
 38. A method of identifying a compound for promoting sprouting of new nerve fibers from blood vessel explants, which method comprises assaying candidate compounds for nerve growth promoting activity ex vivo using the method of claim 36, and identifying a compound that promotes nerve growth in a blood vessel explant as indicative of a compound that promotes nerve growth.
 39. A method of inducing angiogenesis, which method comprises the steps of: a) co-culturing an isolated blood vessel and an isolated muscle explant; and b) incubating with a growth-stimulating compound.
 40. The method of claim 39, wherein the compound is a combination of VEGF and PDGF.
 41. A method of identifying a compound for inducing angiogenesis, which method comprises assaying candidate compounds for angiogenic activity ex vivo using the method of claim 39, and identifying a compound that promotes angiogenesis in a blood vessel cell as indicative of a compound that promotes angiogenesis.
 42. A method for promoting growth of cavernous nerves form major pelvic ganglia (MPG), which method comprises contacting said MPG with an effective amount of a factor, wherein the factor is selected from the group consisting of vascular endothelial growth factor (VEGF), brain-derived growth factor (BDNF), basic fibroblast growth factor (bFGF), neurotrophin-3 (NT-3), neurotrophin-4 (NT-4), platelet-derived growth factor (PDGF), angiopoietin-1 (Ang-1), thereby promoting growth of said cavernous nerves form said MPG.
 43. A method of identifying a compound that promotes growth of cavernous nerves from major pelvic ganglia (MPG), which method comprises: a) in vitro culturing MPG; b) measuring growth of cavernous nerves from said MPG in the presence and absence of a candidate compound; and c) identifying a compound that promotes nerve growth as indicative of a compound that promotes growth of cavernous nerves from said MPG. 